Stitched polypeptides

ABSTRACT

The present invention provides inventive stitched polypeptides, pharmaceutical compositions thereof, and methods of making and using inventive stitched polypeptides.

PRIORITY INFORMATION

The present application is a divisional of and claims priority under 35U.S.C. § 120 to U.S. patent application Ser. No. 14/027,064, filed Sep.13, 2013, which is a divisional of and claims priority under 35 U.S.C. §120 to U.S. patent application Ser. No. 12/593,384, filed Mar. 5, 2010,which is a national stage filing under 35 U.S.C. § 371 of internationalPCT application, PCT/US2008/058575, filed Mar. 28, 2008, which claimspriority under 35 U.S.C. § 119(e) to U.S. provisional patent applicationSer. No. 60/908,566, filed Mar. 28, 2007, each of which is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

The important biological roles that peptides and polypeptides play ashormones, enzyme inhibitors, substrates, neurotransmitters, andneuromediators has led to the widespread use of peptides or peptidemimetics in medicinal chemistry as therapeutic agents. The peptide'sbioactive conformation, combining structural elements such as alphahelices, beta sheets, turns, and/or loops, is important as it allows forselective biological recognition of receptors or enzymes, therebyinfluencing cell-cell communication and/or controlling vital cellfunctions, such as metabolism, immune defense, and reproduction (Babineet al., Chem. Rev. (1997) 97:1359). The alpha-helix is one of the majorstructural components of peptides. However, alpha-helical peptides havea propensity for unraveling and forming random coils, which are, in mostcases, biologically less active, or even inactive, and are highlysusceptible to proteolytic degradation.

Many research groups have developed strategies for the design andsynthesis of more robust peptides as therapeutics. For example, onestrategy has been to incorporate more robust functionalities into thepeptide chain while still maintaining the peptide's unique conformationand secondary structure (see, for example, Gante, J. Angew. Chem. Int.Ed. Engl. (1994) 33:1699-1720; R. M. J. Liskamp, Recl. Tray. Chim.Pays-Bas 1994, 113, 1; Giannis, T. Kolter, Angew. Chem. Int. Ed. Engl.1993, 32, 1244; P. D. Bailey, Peptide Chemistry, Wiley, New York, 1990,p. 182; and references cited therein). Another approach has been tostabilize the peptide via covalent cross-links (see, for example, Phelanet al. 1997 J. Am. Chem. Soc. 119:455; Leuc et al. 2003 Proc. Nat'l.Acad. Sci. USA 100:11273; Bracken et al., 1994 J. Am. Chem. Soc.116:6432; Yan et al. 2004 Bioorg. Med. Chem. 14:1403). However, themajority of the reported methodologies involve use of polar and/orlabile crosslinking groups.

SUMMARY OF THE INVENTION

“Peptide stapling” is a term coined from a synthetic methodology whereintwo olefin-containing sidechains present in a polypeptide chain arecovalently joined (e.g., “stapled together”) using a ring-closingmetathesis (RCM) reaction to form a cross-linked ring (see, the coverart for J. Org. Chem. (2001) vol. 66, issue 16 describingmetathesis-based crosslinking of alpha-helical peptides; Blackwell etal.; Angew Chem. Int. Ed. (1994) 37:3281). However, the term “peptidestapling,” as used herein, encompasses the joining of two doublebond-containing sidechains, two triple bond-containing sidechains, orone double bond-containing and one triple bond-containing side chain,which may be present in a polypeptide chain, using any number ofreaction conditions and/or catalysts to facilitate such a reaction, toprovide a singly “stapled” polypeptide. Additionally, the term “peptidestitching,” as used herein, refers to multiple and tandem “stapling”events in a single polypeptide chain to provide a “stitched” (multiplystapled) polypeptide.

Stapling of a peptide using all-hydrocarbon cross-link has been shown tohelp maintain its native conformation and/or secondary structure,particularly under physiologically relevant conditions (seeSchafmiester, et al., J. Am. Chem. Soc. (2000) 122:5891-5892; Walenskyet al., Science (2004) 305:1466-1470). For example, stapling apolypeptide by an all-hydrocarbon crosslink predisposed to have analpha-helical secondary structure can constrain the polypeptide to itsnative alpha-helical conformation. The constrained secondary structuremay, for example, increase the peptide's resistance to proteolyticcleavage, may increase the peptide's hydrophobicity, may allow forbetter penetration of the peptide into the target cell's membrane (e.g.,through an energy-dependent transport mechanism such as pinocytosis),and/or may lead to an improvement in the peptide's biological activityrelative to the corresponding uncrosslinked (e.g., “unstitched” or“unstapled”) peptide. Such constraints have been applied to theapoptosis-inducing BID-BH3 alpha-helix, resulting in a highersuppression of malignant growth of leukemia in an animal model comparedto the unstitched polypeptide; see Walensky et al., Science (2004)305:1466-1470; U.S. Patent Application Publication No. 2005/02506890;and U.S. Patent Application Publication No. 2006/0008848, each of whichis incorporated herein by reference.

Novel stitched polypeptides and their “unstitched” precursors are thefocus of the present invention. The present invention provides novelstitched and “unstitched” polypeptides, and methods for theirpreparation and use. The present invention also provides pharmaceuticalcompositions, including pharmaceutical compositions for oraladministration, comprising an inventive stitched polypeptide and apharmaceutically acceptable excipient. In certain embodiments, thepresent invention provides novel alpha-helical stitched polypeptides. Incertain embodiments, the inventive alpha-helical polypeptides retaintheir alpha-helical structure under physiological conditions, such as inthe body of a subject (e.g., in the gastrointestinal tract; in thebloodstream).

Thus, in certain embodiments, the present invention provides an“unstitched” substantially alpha-helical polypeptide of the formula:

wherein:

each instance of K, L₁, L₂, and M, is, independently, a bond, cyclic oracyclic, branched or unbranched, substituted or unsubstituted alkylene;cyclic or acyclic, branched or unbranched, substituted or unsubstitutedalkenylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted alkynylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroalkylene; cyclic or acyclic, branchedor unbranched, substituted or unsubstituted heteroalkenylene; cyclic oracyclic, branched or unbranched, substituted or unsubstitutedheteroalkynylene; substituted or unsubstituted arylene; substituted orunsubstituted heteroarylene; or substituted or unsubstituted acylene;

each instance of R^(a) is, independently, hydrogen; cyclic or acyclic,branched or unbranched, substituted or unsubstituted aliphatic; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; cyclic or acyclic, substituted orunsubstituted acyl; or R^(a) is a suitable amino protecting group;

each instance of R^(b) is, independently, a suitable amino acid sidechain; hydrogen; cyclic or acyclic, branched or unbranched, substitutedor unsubstituted aliphatic; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; cyclic oracyclic, substituted or unsubstituted acyl; substituted or unsubstitutedhydroxyl; substituted or unsubstituted thiol; substituted orunsubstituted amino; cyano; isocyano; halo; or nitro;

each instance of R^(c), is, independently, hydrogen; cyclic or acyclic,branched or unbranched, substituted or unsubstituted aliphatic; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; cyclic or acyclic, substituted orunsubstituted acyl; substituted or unsubstituted hydroxyl; substitutedor unsubstituted thiol; substituted or unsubstituted amino; cyano;isocyano; halo; or nitro;

each instance of R^(e) is, independently, —R^(E), —OR^(E), —N(R^(E))₂,or —SR^(E), wherein each instance of R^(E) is, independently, hydrogen,cyclic or acyclic, branched or unbranched, substituted or unsubstitutedaliphatic; cyclic or acyclic, branched or unbranched, substituted orunsubstituted heteroaliphatic; substituted or unsubstituted aryl;substituted or unsubstituted heteroaryl; substituted or unsubstitutedacyl; a resin; a suitable hydroxyl, amino or thiol protecting group; ortwo R^(E) groups together form a substituted or unsubstituted 5- to6-membered heterocyclic or heteroaromatic ring;

each instance of R^(f) is, independently, hydrogen, cyclic or acyclic,branched or unbranched, substituted or unsubstituted aliphatic; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; substituted or unsubstituted acyl; a resin; asuitable amino protecting group; a label optionally joined by a linker,wherein the linker is selected from cyclic or acyclic, branched orunbranched, substituted or unsubstituted alkylene; cyclic or acyclic,branched or unbranched, substituted or unsubstituted alkenylene; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedalkynylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted heteroalkylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroalkenylene; cyclic or acyclic,branched or unbranched, substituted or unsubstituted heteroalkynylene;substituted or unsubstituted arylene; substituted or unsubstitutedheteroarylene; or substituted or unsubstituted acylene; or R^(f) andR^(a) together form a substituted or unsubstituted 5- to 6-memberedheterocyclic or heteroaromatic ring;

each instance of X_(AA) is, independently, a natural or unnatural aminoacid;

each instance of x is, independently, an integer between 0 to 3;

y and z are, independently, an integer between 2 to 6;

j is, independently, an integer between 1 to 10;

p is an integer between 0 to 10;

each instance of s and t is, independently, an integer between 0 and100; and

wherein

corresponds to a double or triple bond.

The amino acid sequence of the peptide may be substantially similar toor homologous to a known bioactive peptide.

In certain embodiments, the present invention provides a “stitched”substantially alpha-helical polypeptide of the formula:

wherein

K, L₁, L₂, M, R^(a), R^(b), R^(e), R^(f), s, t, y, z, j, p, and X_(AA)are as defined herein;

each instance of R^(KL), R^(LL), and R^(LM), is, independently,hydrogen; cyclic or acyclic, branched or unbranched, substituted orunsubstituted aliphatic; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; substitutedor unsubstituted acyl; substituted or unsubstituted hydroxyl;substituted or unsubstituted thiol; substituted or unsubstituted amino;azido; cyano; isocyano; halo; nitro;

or two adjacent R^(KL) groups are joined to form a substituted orunsubstituted 5- to 8-membered cycloaliphatic ring; substituted orunsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted orunsubstituted aryl ring; or substituted or unsubstituted heteroarylring; two adjacent R^(KL) groups are joined to form a substituted orunsubstituted 5- to 8-membered cycloaliphatic ring; substituted orunsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted orunsubstituted aryl ring; or substituted or unsubstituted heteroarylring; or two adjacent R^(LM) groups are joined to form a substituted orunsubstituted 5- to 8-membered cycloaliphatic ring; substituted orunsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted orunsubstituted aryl ring; or substituted or unsubstituted heteroarylring;

each instance of u, v, and q, is, independently, an integer between 0 to4; and

corresponds to a single, double, or triple bond.

In certain embodiments, the present invention also providessubstantially alpha-helical polypeptides of the formulae:

wherein K, L₁, L₂, M, R^(a), R^(b), R^(e), R^(f), R^(c), R^(KL), R^(LL),R^(LM), s, t, x, y, z, j, p, v, u, q, X_(AA),

and

are defined herein.

The present invention is also directed to a method of making asubstantially alpha-helical polypeptide, said method comprising thesteps of:

(i) providing a bis-amino acid of the formula (A):

wherein L₁, L₂, R^(a), R^(e), R^(f), R^(c), x, and

are defined herein;

(ii) providing an amino acid of the formula (B):

wherein K, R^(a), R^(b), R^(e), R^(f), R^(c), x, and

are defined herein;

(iii) providing an amino acid of the formula (C):

wherein M, R^(a), R^(b), R^(e), R^(f), R^(c), x, and

are defined herein;

(iv) providing at least one additional amino acid; and

(v) reacting said amino acids of formulae (A), (B), and (C) with atleast one amino acid of step (iv) to provide a polypeptide of formula(I).

In certain embodiments, the above method further comprises making asubstantially alpha-helical polypeptide of formulae (II) to (VII) by(vi) treating the polypeptide of step (v) with a catalyst. In certainembodiments, the catalyst is a ring closing metathesis catalyst.

The present invention also provides a bis-amino acid having the formula:

wherein L₁, L₂, R^(a), R^(e), R^(f), R^(c), x, and

are defined herein.

Furthermore, the present invention provides a pharmaceutical compositioncomprising a substantially alpha-helical inventive polypeptide and apharmaceutically acceptable excipient.

In certain embodiments, the pharmaceutical composition is suitable fororal administration. In certain embodiments, the pharmaceuticalcomposition is suitable for IV administration.

The present invention is also directed to a method of treating adisease, disorder, or condition in a subject by administering atherapeutically effective amount of a substantially alpha-helicalpolypeptide formulae (II) to (VII) to a subject in need thereof.

This application refers to various issued patent, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference.

The details of one or more embodiments of the invention are set forthherein. Other features, objects, and advantages of the invention will beapparent from the description, the figures, the examples, and theclaims.

DEFINITIONS

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this invention, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th)Ed., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in Organic Chemistry, Thomas Sorrell, University ScienceBooks, Sausalito, 1999; Smith and March March's Advanced OrganicChemistry, 5^(th) Edition, John Wiley & Sons, Inc., New York, 2001;Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., NewYork, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3^(rd)Edition, Cambridge University Press, Cambridge, 1987.

The compounds of the present invention (e.g., amino acids, andunstitched, partially stitched, and stitched peptides and polypeptides)may exist in particular geometric or stereoisomeric forms. The presentinvention contemplates all such compounds, including cis- andtrans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers,(L)-isomers, the racemic mixtures thereof, and other mixtures thereof,as falling within the scope of the invention.

Where an isomer/enantiomer is preferred, it may, in some embodiments, beprovided substantially free of the corresponding enantiomer, and mayalso be referred to as “optically enriched.” “Optically enriched,” asused herein, means that the compound is made up of a significantlygreater proportion of one enantiomer. In certain embodiments thecompound of the present invention is made up of at least about 90% byweight of a preferred enantiomer. In other embodiments the compound ismade up of at least about 95%, 98%, or 99% by weight of a preferredenantiomer. Preferred enantiomers may be isolated from racemic mixturesby any method known to those skilled in the art, including chiral highpressure liquid chromatography (HPLC) and the formation andcrystallization of chiral salts or prepared by asymmetric syntheses.See, for example, Jacques, et al., Enantiomers, Racemates andResolutions (Wiley Interscience, New York, 1981); Wilen, S. H., et al.,Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistry of CarbonCompounds (McGraw-Hill, N Y, 1962); Wilen, S. H. Tables of ResolvingAgents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of NotreDame Press, Notre Dame, Ind. 1972).

It will be appreciated that the compounds of the present invention, asdescribed herein, may be substituted with any number of substituents orfunctional moieties. In general, the term “substituted” whether precededby the term “optionally” or not, and substituents contained in formulasof this invention, refer to the replacement of hydrogen radicals in agiven structure with the radical of a specified substituent. When morethan one position in any given structure may be substituted with morethan one substituent selected from a specified group, the substituentmay be either the same or different at every position. As used herein,the term “substituted” is contemplated to include substitution with allpermissible substituents of organic compounds, any of the substituentsdescribed herein (for example, aliphatic, alkyl, alkenyl, alkynyl,heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino,thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo,etc.), and any combination thereof (for example, aliphaticamino,heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino,heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy,alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy,heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,heteroarylthioxy, acyloxy, and the like) that results in the formationof a stable moiety. The present invention contemplates any and all suchcombinations in order to arrive at a stable substituent/moiety.Additional examples of generally applicable substitutents areillustrated by the specific embodiments shown in the Examples, which aredescribed herein. For purposes of this invention, heteroatoms such asnitrogen may have hydrogen substituents and/or any suitable substituentas described herein which satisfy the valencies of the heteroatoms andresults in the formation of a stable moiety.

As used herein, substituent names which end in the suffix “-ene” referto a biradical derived from the removal of two hydrogen atoms from thesubstitutent. Thus, for example, acyl is acylene; alkyl is alkylene;alkeneyl is alkenylene; alkynyl is alkynylene; heteroalkyl isheteroalkylene, heteroalkenyl is heteroalkenylene, heteroalkynyl isheteroalkynylene, aryl is arylene, and heteroaryl is heteroarylene.

The term “acyl,” as used herein, refers to a group having the generalformula —C(═O)R^(A), —C(═O)OR^(A), —C(═O)—O—C(═O)R^(A), —C(═O)SR^(A),—C(═O)N(R^(A))₂, —C(═S)R^(A), —C(═S)N(R^(A))₂, and —C(═S)S(R^(A)),—C(═NR^(A))R^(A), —C(═NR^(A))OR^(A), —C(═NR^(A))SR^(A), and—C(═NR^(A))N(R^(A))₂, wherein R^(A) is hydrogen; halogen; substituted orunsubstituted hydroxyl; substituted or unsubstituted thiol; substitutedor unsubstituted amino; substituted or unsubstituted acyl, cyclic oracyclic, substituted or unsubstituted, branched or unbranched aliphatic;cyclic or acyclic, substituted or unsubstituted, branched or unbranchedheteroaliphatic; cyclic or acyclic, substituted or unsubstituted,branched or unbranched alkyl; cyclic or acyclic, substituted orunsubstituted, branched or unbranched alkenyl; substituted orunsubstituted alkynyl; substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkyloxy,heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy,heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,heteroarylthioxy, mono- or di-aliphaticamino, mono- ordi-heteroaliphaticamino, mono- or di-alkylamino, mono- ordi-heteroalkylamino, mono- or di-arylamino, or mono- ordi-heteroarylamino; or two R^(A) groups taken together form a 5- to6-membered heterocyclic ring. Exemplary acyl groups include aldehydes(—CHO), carboxylic acids (—CO₂H), ketones, acyl halides, esters, amides,imines, carbonates, carbamates, and ureas. Acyl substituents include,but are not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety (e.g., aliphatic, alkyl,alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl,oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl,thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino,heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl,aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy,heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy,heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like,each of which may or may not be further substituted).

The term “acyloxy” refers to a “substituted hydroxyl” of the formula(—OR^(i)), wherein R^(i) is an optionally substituted acyl group, asdefined herein, and the oxygen moiety is directly attached to the parentmolecule.

The term “acylene,” as used herein, refers to an acyl group having thegeneral formulae: —R⁰—(C═X¹)—R⁰, —R⁰—X²(C═X¹)—R⁰—, or —R—X²(C═X¹)X³—R⁰—,where X¹, X², and X³ is, independently, oxygen, sulfur, or NR^(r),wherein R^(r) is hydrogen or aliphatic, and R₀ is an optionallysubstituted alkylene, alkenylene, alkynylene, heteroalkylene,heteroalkenylene, or heteroalkynylene group, as defined herein.Exemplary acylene groups wherein R⁰ is alkylene includes—(CH₂)_(T)—O(C═O)—(CH₂)_(T)—; —(CH₂)_(T)—NR^(r)(C═O)—(CH₂)_(T)—;—(CH₂)_(T)—O(C═NR^(r))—(CH₂)_(T)—;—(CH₂)_(T)—NR^(r)(C═NR^(r))—(CH₂)_(T)—; —(CH₂)_(T)—(C═O)—(CH₂)_(T)—;—(CH₂)_(T)—(C═NR^(r))—(CH₂)_(T)—; —(CH₂)_(T)—S(C═S)—(CH₂)_(T)—;—(CH₂)_(T)—NR^(r)(C═S)—(CH₂)_(T)—; —(CH₂)_(T)—S(C═NR^(r))—(CH₂)_(T);—(CH₂)_(T)—O(C═S)—(CH₂)_(T)—; —(CH₂)_(T)—(C═S)—(CH₂)_(T)—; or—(CH₂)_(T)—S(C═O)—(CH₂)_(T)—, and the like, which may bear one or moresubstituents; and wherein each instance of xx is, independently, aninteger between 0 to 20. Acylene groups may be cyclic or acyclic,branched or unbranched, substituted or unsubstituted. Acylenesubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).

The term “aliphatic,” as used herein, includes both saturated andunsaturated, nonaromatic, straight chain (i.e., unbranched), branched,acyclic, and cyclic (i.e., carbocyclic) hydrocarbons, which areoptionally substituted with one or more functional groups. As will beappreciated by one of ordinary skill in the art, “aliphatic” is intendedherein to include, but is not limited to, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as usedherein, the term “alkyl” includes straight, branched and cyclic alkylgroups. An analogous convention applies to other generic terms such as“alkenyl”, “alkynyl”, and the like. Furthermore, as used herein, theterms “alkyl”, “alkenyl”, “alkynyl”, and the like encompass bothsubstituted and unsubstituted groups. In certain embodiments, as usedherein, “aliphatic” is used to indicate those aliphatic groups (cyclic,acyclic, substituted, unsubstituted, branched or unbranched) having 1-20carbon atoms. Aliphatic group substituents include, but are not limitedto, any of the substituents described herein, that result in theformation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl,heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino,thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo,aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino,arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy,heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy,aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy,arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which mayor may not be further substituted).

The term “alkyl,” as used herein, refers to saturated, straight- orbranched-chain hydrocarbon radicals derived from a hydrocarbon moietycontaining between one and twenty carbon atoms by removal of a singlehydrogen atom. In some embodiments, the alkyl group employed in theinvention contains 1-20 carbon atoms. In another embodiment, the alkylgroup employed contains 1-15 carbon atoms. In another embodiment, thealkyl group employed contains 1-10 carbon atoms. In another embodiment,the alkyl group employed contains 1-8 carbon atoms. In anotherembodiment, the alkyl group employed contains 1-5 carbon atoms. Examplesof alkyl radicals include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl,iso-pentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl,n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like, which maybear one or more substitutents. Alkyl group substituents include, butare not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety (e.g., aliphatic, alkyl,alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl,oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl,thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino,heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl,aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy,heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy,heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like,each of which may or may not be further substituted).

The term “alkylene,” as used herein, refers to a biradical derived froman alkyl group, as defined herein, by removal of two hydrogen atoms.Alkylene groups may be cyclic or acyclic, branched or unbranched,substituted or unsubstituted. Alkylene group substituents include, butare not limited to, any of the substituents described herein, thatresult in the formation of a stable moiety (e.g., aliphatic, alkyl,alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl,oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl,thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino,heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl,aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy,heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy,heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like,each of which may or may not be further substituted).

The term “alkenyl,” as used herein, denotes a monovalent group derivedfrom a straight- or branched-chain hydrocarbon moiety having at leastone carbon-carbon double bond by the removal of a single hydrogen atom.In certain embodiments, the alkenyl group employed in the inventioncontains 2-20 carbon atoms. In some embodiments, the alkenyl groupemployed in the invention contains 2-15 carbon atoms. In anotherembodiment, the alkenyl group employed contains 2-10 carbon atoms. Instill other embodiments, the alkenyl group contains 2-8 carbon atoms. Inyet another embodiments, the alkenyl group contains 2-5 carbons. Alkenylgroups include, for example, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, and the like, which may bear one or moresubstituents. Alkenyl group substituents include, but are not limitedto, any of the substituents described herein, that result in theformation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl,heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino,thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo,aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino,arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy,heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy,aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy,arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which mayor may not be further substituted).

The term “alkenylene,” as used herein, refers to a biradical derivedfrom an alkenyl group, as defined herein, by removal of two hydrogenatoms. Alkenylene groups may be cyclic or acyclic, branched orunbranched, substituted or unsubstituted. Alkenylene group substituentsinclude, but are not limited to, any of the substituents describedherein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).

The term “alkynyl,” as used herein, refers to a monovalent group derivedfrom a straight- or branched-chain hydrocarbon having at least onecarbon-carbon triple bond by the removal of a single hydrogen atom. Incertain embodiments, the alkynyl group employed in the inventioncontains 2-20 carbon atoms. In some embodiments, the alkynyl groupemployed in the invention contains 2-15 carbon atoms. In anotherembodiment, the alkynyl group employed contains 2-10 carbon atoms. Instill other embodiments, the alkynyl group contains 2-8 carbon atoms. Instill other embodiments, the alkynyl group contains 2-5 carbon atoms.Representative alkynyl groups include, but are not limited to, ethynyl,2-propynyl (propargyl), 1-propynyl, and the like, which may bear one ormore substituents. Alkynyl group substituents include, but are notlimited to, any of the substituents described herein, that result in theformation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl,heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino,thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo,aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino,arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy,heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy,aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy,arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which mayor may not be further substituted).

The term “alkynylene,” as used herein, refers to a biradical derivedfrom an alkynylene group, as defined herein, by removal of two hydrogenatoms. Alkynylene groups may be cyclic or acyclic, branched orunbranched, substituted or unsubstituted. Alkynylene group substituentsinclude, but are not limited to, any of the substituents describedherein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).

The term “amino,” as used herein, refers to a group of the formula(—NH₂). A “substituted amino” refers either to a mono-substituted amine(—NHR^(h)) of a disubstituted amine (—NR^(h) ₂), wherein the R^(h)substituent is any substitutent as described herein that results in theformation of a stable moiety (e.g., a suitable amino protecting group;aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, amino, nitro, hydroxyl, thiol, halo, aliphaticamino,heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino,heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy,alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy,heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,heteroarylthioxy, acyloxy, and the like, each of which may or may not befurther substituted). In certain embodiments, the R^(h) substituents ofthe di-substituted amino group (—NR^(h) ₂) form a 5- to 6-memberedheterocyclic ring.

The term “aliphaticamino,” refers to a “substituted amino” of theformula (—NR^(h) ₂), wherein R^(h) is, independently, a hydrogen or anoptionally substituted aliphatic group, as defined herein, and the aminomoiety is directly attached to the parent molecule.

The term “aliphaticoxy,” refers to a “substituted hydroxyl” of theformula (—OR^(i)), wherein R^(i) is an optionally substituted aliphaticgroup, as defined herein, and the oxygen moiety is directly attached tothe parent molecule.

The term “alkyloxy” refers to a “substituted hydroxyl” of the formula(—OR^(i)), wherein R^(i) is an optionally substituted alkyl group, asdefined herein, and the oxygen moiety is directly attached to the parentmolecule.

The term “alkylthioxy” refers to a “substituted thiol” of the formula(—SR^(r)), wherein R^(r) is an optionally substituted alkyl group, asdefined herein, and the sulfur moiety is directly attached to the parentmolecule.

The term “alkylamino” refers to a “substituted amino” of the formula(—NR^(h) ₂), wherein R^(h) is, independently, a hydrogen or anoptionally substituted alkyl group, as defined herein, and the nitrogenmoiety is directly attached to the parent molecule.

The term “aryl,” as used herein, refer to stable aromatic mono- orpolycyclic ring system having 3-20 ring atoms, of which all the ringatoms are carbon, and which may be substituted or unsubstituted. Incertain embodiments of the present invention, “aryl” refers to a mono,bi, or tricyclic C₄-C₂₀ aromatic ring system having one, two, or threearomatic rings which include, but not limited to, phenyl, biphenyl,naphthyl, and the like, which may bear one or more substituents. Arylsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).

The term “arylene,” as used herein refers to an aryl biradical derivedfrom an aryl group, as defined herein, by removal of two hydrogen atoms.Arylene groups may be substituted or unsubstituted. Arylene groupsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).Additionally, arylene groups may be incorporated as a linker group intoan alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene,or heteroalkynylene group, as defined herein.

The term “arylalkyl,” as used herein, refers to an aryl substitutedalkyl group, wherein the terms “aryl” and “alkyl” are defined herein,and wherein the aryl group is attached to the alkyl group, which in turnis attached to the parent molecule. An exemplary arylalkyl groupincludes benzyl.

The term “aryloxy” refers to a “substituted hydroxyl” of the formula(—OR^(i)), wherein R^(i) is an optionally substituted aryl group, asdefined herein, and the oxygen moiety is directly attached to the parentmolecule.

The term “arylamino,” refers to a “substituted amino” of the formula(—NR^(h) ₂), wherein R^(h) is, independently, a hydrogen or anoptionally substituted aryl group, as defined herein, and the nitrogenmoiety is directly attached to the parent molecule.

The term “arylthioxy” refers to a “substituted thiol” of the formula(—SR^(r)), wherein R^(r) is an optionally substituted aryl group, asdefined herein, and the sulfur moiety is directly attached to the parentmolecule.

The term “azido,” as used herein, refers to a group of the formula(—N₃). An “optionally substituted azido” refers to a group of theformula (—N₃R^(t)), wherein R^(t) can be any substitutent (other thanhydrogen). Substituents include, but are not limited to, any of thesubstituents described herein, that result in the formation of a stablemoiety (e.g., a suitable amino protecting group; (e.g., aliphatic,alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, cyano, amino, nitro, hydroxyl, aliphaticamino,heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino,heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy,alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy,heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,heteroarylthioxy, and the like, each of which may or may not be furthersubstituted).

The term “cyano,” as used herein, refers to a group of the formula(—CN).

The terms “halo” and “halogen” as used herein refer to an atom selectedfrom fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo,—Br), and iodine (iodo, —I).

The term “heteroaliphatic,” as used herein, refers to an aliphaticmoiety, as defined herein, which includes both saturated andunsaturated, nonaromatic, straight chain (i.e., unbranched), branched,acyclic, cyclic (i.e., heterocyclic), or polycyclic hydrocarbons, whichare optionally substituted with one or more functional groups, and thatcontain one or more oxygen, sulfur, nitrogen, phosphorus, or siliconatoms, e.g., in place of carbon atoms. In certain embodiments,heteroaliphatic moieties are substituted by independent replacement ofone or more of the hydrogen atoms thereon with one or more substituents.As will be appreciated by one of ordinary skill in the art,“heteroaliphatic” is intended herein to include, but is not limited to,heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl,heterocycloalkenyl, and heterocycloalkynyl moieties. Thus, the term“heteroaliphatic” includes the terms “heteroalkyl,” “heteroalkenyl”,“heteroalkynyl”, and the like. Furthermore, as used herein, the terms“heteroalkyl”, “heteroalkenyl”, “heteroalkynyl”, and the like encompassboth substituted and unsubstituted groups. In certain embodiments, asused herein, “heteroaliphatic” is used to indicate those heteroaliphaticgroups (cyclic, acyclic, substituted, unsubstituted, branched orunbranched) having 1-20 carbon atoms. Heteroaliphatic group substituentsinclude, but are not limited to, any of the substituents describedherein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino, thiooxo, cyano,isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino,heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino,heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy,alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy,heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,heteroarylthioxy, acyloxy, and the like, each of which may or may not befurther substituted).

The term “heteroaliphaticamino” refers to a “substituted amino” of theformula (—NR^(h) ₂), wherein R^(h) is, independently, a hydrogen or anoptionally substituted heteroaliphatic group, as defined herein, and thenitrogen moiety is directly attached to the parent molecule.

The term “heteroaliphaticoxy” refers to a “substituted hydroxyl” of theformula (—OR^(i)), wherein R^(i) is an optionally substitutedheteroaliphatic group, as defined herein, and the oxygen moiety isdirectly attached to the parent molecule.

The term “heteroaliphaticthioxy” refers to a “substituted thiol” of theformula (—SR^(r)), wherein R^(r) is an optionally substitutedheteroaliphatic group, as defined herein, and the sulfur moiety isdirectly attached to the parent molecule.

The term “heteroalkyl,” as used herein, refers to an alkyl moiety, asdefined herein, which contain one or more oxygen, sulfur, nitrogen,phosphorus, or silicon atoms, e.g., in place of carbon atoms.

The term “heteroalkylene,” as used herein, refers to a biradical derivedfrom an heteroalkyl group, as defined herein, by removal of two hydrogenatoms. Heteroalkylene groups may be cyclic or acyclic, branched orunbranched, substituted or unsubstituted. Heteroalkylene groupsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).

The term “heteroalkenyl,” as used herein, refers to an alkenyl moiety,as defined herein, which contain one or more oxygen, sulfur, nitrogen,phosphorus, or silicon atoms, e.g., in place of carbon atoms.l

The term “heteroalkenylene,” as used herein, refers to a biradicalderived from an heteroalkenyl group, as defined herein, by removal oftwo hydrogen atoms. Heteroalkenylene groups may be cyclic or acyclic,branched or unbranched, substituted or unsubstituted.

The term “heteroalkynyl,” as used herein, refers to an alkynyl moiety,as defined herein, which contain one or more oxygen, sulfur, nitrogen,phosphorus, or silicon atoms, e.g., in place of carbon atoms.

The term “heteroalkynylene,” as used herein, refers to a biradicalderived from an heteroalkynyl group, as defined herein, by removal oftwo hydrogen atoms. Heteroalkynylene groups may be cyclic or acyclic,branched or unbranched, substituted or unsubstituted.

The term “heteroalkylamino” refers to a “substituted amino” of theformula (—NR^(h) ₂), wherein R^(h) is, independently, a hydrogen or anoptionally substituted heteroalkyl group, as defined herein, and thenitrogen moiety is directly attached to the parent molecule.

The term “heteroalkyloxy” refers to a “substituted hydroxyl” of theformula (—OR^(i)), wherein R^(i) is an optionally substitutedheteroalkyl group, as defined herein, and the oxygen moiety is directlyattached to the parent molecule.

The term “heteroalkylthioxy” refers to a “substituted thiol” of theformula (—SR^(r)), wherein R^(r) is an optionally substitutedheteroalkyl group, as defined herein, and the sulfur moiety is directlyattached to the parent molecule.

The term “heterocyclic,” “heterocycles,” or “heterocyclyl,” as usedherein, refers to a cyclic heteroaliphatic group. A heterocyclic grouprefers to a non-aromatic, partially unsaturated or fully saturated, 3-to 10-membered ring system, which includes single rings of 3 to 8 atomsin size, and bi- and tri-cyclic ring systems which may include aromaticfive- or six-membered aryl or heteroaryl groups fused to a non-aromaticring. These heterocyclic rings include those having from one to threeheteroatoms independently selected from oxygen, sulfur, and nitrogen, inwhich the nitrogen and sulfur heteroatoms may optionally be oxidized andthe nitrogen heteroatom may optionally be quaternized. In certainembodiments, the term heterocylic refers to a non-aromatic 5-, 6-, or7-membered ring or polycyclic group wherein at least one ring atom is aheteroatom selected from O, S, and N (wherein the nitrogen and sulfurheteroatoms may be optionally oxidized), and the remaining ring atomsare carbon, the radical being joined to the rest of the molecule via anyof the ring atoms. Heterocycyl groups include, but are not limited to, abi- or tri-cyclic group, comprising fused five, six, or seven-memberedrings having between one and three heteroatoms independently selectedfrom the oxygen, sulfur, and nitrogen, wherein (i) each 5-membered ringhas 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds,and each 7-membered ring has 0 to 3 double bonds, (ii) the nitrogen andsulfur heteroatoms may be optionally oxidized, (iii) the nitrogenheteroatom may optionally be quaternized, and (iv) any of the aboveheterocyclic rings may be fused to an aryl or heteroaryl ring. Exemplaryheterocycles include azacyclopropanyl, azacyclobutanyl,1,3-diazatidinyl, piperidinyl, piperazinyl, azocanyl, thiaranyl,thietanyl, tetrahydrothiophenyl, dithiolanyl, thiacyclohexanyl,oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropuranyl, dioxanyl,oxathiolanyl, morpholinyl, thioxanyl, tetrahydronaphthyl, and the like,which may bear one or more substituents. Substituents include, but arenot limited to, any of the substituents described herein, that result inthe formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl,alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl,sulfinyl, sulfonyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).

The term “heteroaryl,” as used herein, refer to stable aromatic mono- orpolycyclic ring system having 3-20 ring atoms, of which one ring atom isselected from S, O, and N; zero, one, or two ring atoms are additionalheteroatoms independently selected from S, O, and N; and the remainingring atoms are carbon, the radical being joined to the rest of themolecule via any of the ring atoms. Exemplary heteroaryls include, butare not limited to pyrrolyl, pyrazolyl, imidazolyl, pyridinyl,pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl,pyyrolizinyl, indolyl, quinolinyl, isoquinolinyl, benzoimidazolyl,indazolyl, quinolinyl, isoquinolinyl, quinolizinyl, cinnolinyl,quinazolynyl, phthalazinyl, naphthridinyl, quinoxalinyl, thiophenyl,thianaphthenyl, furanyl, benzofuranyl, benzothiazolyl, thiazolynyl,isothiazolyl, thiadiazolynyl, oxazolyl, isoxazolyl, oxadiaziolyl,oxadiaziolyl, and the like, which may bear one or more substituents.Heteroaryl substituents include, but are not limited to, any of thesubstituents described herein, that result in the formation of a stablemoiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic,heterocyclic, aryl, heteroaryl, acyl, sulfinyl, sulfonyl, oxo, imino,thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo,aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino,arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy,heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy,aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy,arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which mayor may not be further substituted).

The term “heteroarylene,” as used herein, refers to a biradical derivedfrom an heteroaryl group, as defined herein, by removal of two hydrogenatoms. Heteroarylene groups may be substituted or unsubstituted.Additionally, heteroarylene groups may be incorporated as a linker groupinto an alkylene, alkenylene, alkynylene, heteroalkylene,heteroalkenylene, or heteroalkynylene group, as defined herein.Heteroarylene group substituents include, but are not limited to, any ofthe substituents described herein, that result in the formation of astable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl,heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino,thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo,aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino,arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy,heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy,aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy,arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which mayor may not be further substituted).

The term “heteroarylamino” refers to a “substituted amino” of the(—NR^(h) ₂), wherein R^(h) is, independently, a hydrogen or anoptionally substituted heteroaryl group, as defined herein, and thenitrogen moiety is directly attached to the parent molecule.

The term “heteroaryloxy” refers to a “substituted hydroxyl” of theformula (—OR^(i)), wherein R^(i) is an optionally substituted heteroarylgroup, as defined herein, and the oxygen moiety is directly attached tothe parent molecule.

The term “heteroarylthioxy” refers to a “substituted thiol” of theformula (—SR^(r)), wherein R^(r) is an optionally substituted heteroarylgroup, as defined herein, and the sulfur moiety is directly attached tothe parent molecule.

The term “hydroxy,” or “hydroxyl,” as used herein, refers to a group ofthe formula (—OH). A “substituted hydroxyl” refers to a group of theformula (—OR^(i)), wherein R^(i) can be any substitutent which resultsin a stable moiety (e.g., a suitable hydroxyl protecting group;aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, nitro, alkylaryl, arylalkyl, and the like, each ofwhich may or may not be further substituted).

The term “imino,” as used herein, refers to a group of the formula(═NR^(r)), wherein R^(r) corresponds to hydrogen or any substitutent asdescribed herein, that results in the formation of a stable moiety (forexample, a suitable amino protecting group; aliphatic, alkyl, alkenyl,alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, amino,hydroxyl, alkylaryl, arylalkyl, and the like, each of which may or maynot be further substituted).

The term “isocyano,” as used herein, refers to a group of the formula(—NC).

The term “nitro,” as used herein, refers to a group of the formula(—NO₂).

The term “oxo,” as used herein, refers to a group of the formula (═O).

As used herein, the term “resin” refers to a resin useful for solidphase synthesis. Solid phase synthesis is a well-known synthetictechnique; see generally, Atherton, E., Sheppard, R. C. Solid PhasePeptide Synthesis: A Practical Approach, IRL Press, Oxford, England,1989, and Stewart J. M., Young, J. D. Solid Phase Peptide Synthesis, 2ndedition, Pierce Chemical Company, Rockford, 1984, the entire contents ofeach of which are hereby incorporated herein by reference. Exemplaryresins which may be employed by the present invention include, but arenot limited to:

(1) alkenyl resins (e.g., REM resin, vinyl sulfone polymer-bound resin,vinyl-polystyrene resin);

(2) amine functionalized resins (e.g., amidine resin,N-(4-Benzyloxybenzyl)hydroxylamine polymer bound,(aminomethyl)polystyrene, polymer bound (R)-(+)-a-methylbenzylamine,2-Chlorotrityl Knorr resin, 2-N-Fmoc-Amino-dibenzocyclohepta-1,4-diene,polymer-bound resin,4-[4-(1-Fmoc-aminoethyl)-2-methoxy-5-nitrophenoxy]butyramidomethyl-polystyreneresin, 4-Benzyloxybenzylamine, polymer-bound,4-Carboxybenzenesulfonamide, polymer-bound,Bis(tert-butoxycarbonyl)thiopseudourea, polymer-bound,Dimethylaminomethyl-polystyrene, Fmoc-3-amino-3-(2-nitrophenyl)propionicacid, polymer-bound, N-Methyl aminomethylated polystyrene, PAL resin,Sieber amide resin, tert-Butyl N-(2-mercaptoethyl)carbamate,polymer-bound, Triphenylchloromethane-4-carboxamide polymer bound);

(3) benzhydrylamine (BHA) resins (e.g., 2-Chlorobenzhydryl chloride,polymer-bound, HMPB-benzhydrylamine polymer bound, 4-Methylbenzhydrol,polymer-bound, Benzhydryl chloride, polymer-bound, Benzhydrylaminepolymer-bound);

(4) Br-functionalized resins (e.g., 4-(Benzyloxy)benzyl bromide polymerbound, 4-Bromopolystyrene, Brominated PPOA resin, Brominated Wang resin,Bromoacetal, polymer-bound, Bromopolystyrene, HypoGel® 200 Br,Polystyrene A-Br for peptide synthesis, Selenium bromide, polymer-bound,TentaGel HL-Br, TentaGel MB-Br, TentaGel S-Br, TentaGel S-Br);

(5) Chloromethyl resins (e.g., 5-[4-(Chloromethyl)phenyl]pentyl]styrene,polymer-bound, 4-(Benzyloxy)benzyl chloride polymer bound,4-Methoxybenzhydryl chloride, polymer-bound);

(6) CHO-functionalized resins (e.g.,(4-Formyl-3-methoxyphenoxymethyl)polystyrene,(4-Formyl-3-methoxyphenoxymethyl)polystyrene, 3-Benzyloxybenzaldehyde,polymer-bound, 4-Benzyloxy-2,6-dimethoxybenzaldehyde,polymer-bound,Formylpolystyrene, HypoGel® 200 CHO, Indole resin, PolystyreneA-CH(OEt)₂, TentaGel HL-CH(OEt)₂);

(7) Cl-functionalized resins (e.g., Benzoyl chloride polymer bound,(Chloromethyl)polystyrene, Merrifield's resin);

(8) CO₂H functionalized resins (e.g., Carboxyethylpolystryrene, HypoGel®200 COOH, Polystyrene AM-COOH, TentaGel HL-COOH, TentaGel MB—COOH,TentaGel S—COOH);

(9) Hypo-Gel resins (e.g., HypoGel® 200 FMP, HypoGel® 200 PHB, HypoGel®200 Trt-OH, HypoGel® 200 HMB);

(10) I-functionalized resins (e.g., 4-lodophenol, polymer-bound,lodopolystyrene); Janda-Jels™ (JandaJel^(ä)-Rink amide, JandaJel-NH₂,JandaJel-C1, JandaJel-4-Mercaptophenol, JandaJel-OH,JandaJel-1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide,JandaJel-1,3,4,6,7,8-hexahydro-2H-pyrimido-[1,2-a] pyrimidine,JandaJel-morpholine, JandaJel-polypyridine, JandaJel-Triphenylphosphine,JandaJel-Wang);

(11) MBHA resins (3 [4′-(Hydroxymethyl)phenoxy] propionicacid-4-methylbenzhydrylamine resin, 4-(Hydroxymethyl)phenoxyacetic acidpolymer-bound to MBHA resin, HMBA-4-methylbenzhydrylamine polymer bound,4-Methylbenzhydrylamine hydrochloride polymer bound Capacity (amine));

(12) NH₂ functionalized resins ((Aminomethyl)polystyrene,(Aminomethyl)polystyrene, HypoGel® 200 NH2, Polystyrene AM-NH₂,Polystyrene Microspheres 2-aminoethylated, Polystyrol Microspheres2-bromoethylated, Polystyrol Microspheres 2-hydroxyethylated, TentaGelHL-NH₂, Tentagel M Br, Tentagel M NH₂, Tentagel M OH, TentaGel MB-NH₂,TentaGel S-NH₂, TentaGel S-NH₂);

(13) OH-functionalized resins (e.g., 4-Hydroxymethylbenzoic acid,polymer-bound, Hydroxymethyl Resins, OH-functionalized Wang Resins);

(14) oxime resins (e.g., 4-Chlorobenzophenone oxime polymer bound,Benzophenone oxime polymer bound, 4-Methoxybenzophenone oxime polymerbound);

(15) PEG resins (e.g., ethylene glycol polymer bound);

(16) Boc-/Blz peptide synthesis resins (e.g.,Boc-Lys(Boc)-Lys[Boc-Lys(Boc)]-Cys(Acm)-b-Ala-O-PAM resin,Boc-Lys(Fmoc)-Lys[Boc-Lys(Fmoc)]-b-Ala-O-Pam resin,Boc-Lys(Boc)-Lys[Boc-Lys(Boc)]-Lys{Boc-Lys(Boc)-Lys[Boc-Lys(Boc)]}-b-Ala-O-PAMresin,Boc-Lys(Fmoc)-Lys[Boc-Lys(Fmoc)]-Lys{Boc-Lys(Fmoc)-Lys[Boc-Lys(Fmoc)]}-b-Ala-O-PAMresin, Boc-Lys(Boc)-Lys[Boc-Lys(Boc)]-Lys{Boc-Lys(Boc)-Lys[Boc-Lys(Boc)]}-Cys(Acm)-b-Ala-O-PAM resin, PreloadedPAM resins);

(17) Fmoc-/t-Bu peptide synthesis resins (e.g.,Fmoc-Lys(Fmoc)-Lys[Fmoc-Lys(Fmoc)]-b-Ala-O-Wang resin,Fmoc-Lys(Fmoc)-Lys[Fmoc-Lys(Fmoc)]-Lys{Fmoc-Lys(Fmoc)-Lys[Fmoc-Lys(Fmoc)]}-b-Ala-O-Wang resin, PreloadedTentaGel® S Trityl Resins, Preloaded TentaGel® Resins, Preloaded TritylResins, Preloaded Wang Resins, Trityl Resins Preloaded with AminoAlcohols);

(19) thiol-functionalized resins (e.g., HypoGel® 200 S-Trt, PolystyreneAM-S-Trityl, TentaGel HL-S-Trityl, TentaGel MB-S-Trityl, TentaGelS-S-Trityl); and

(20) Wang resins (e.g., Fmoc-Ala-Wang resin, Fmoc-Arg(Pbf)-Wang resin,Fmoc-Arg(Pmc)-Wang resin, Fmoc-Asn(Trt)-Wang resin, Fmoc-Asp(OtBu)-Wangresin, Fmoc-Cys(Acm)-Wang resin, Fmoc-Cys(StBu)-Wang resin,Fmoc-Cys(Trt) Wang resin, Fmoc-Gln(Trt)-Wang resin, Fmoc-Glu(OtBu)-Wangresin, Fmoc-Gly-Wang resin, Fmoc-His(Trt)-Wang resin, Fmoc-Ile-Wangresin, Fmoc-Leu-Wang resin, Fmoc-Lys(Boc)-Wang resin, Fmoc-Met-Wangresin, Fmoc-D-Met-Wang resin, Fmoc-Phe-Wang resin, Fmoc-Pro-Wang resin,Fmoc-Ser(tBu)-Wang resin, Fmoc-Ser(Trt)-Wang resin, Fmoc-Thr(tBu)-Wangresin, Fmoc-Trp(Boc) Wang resin, Fmoc-Trp-Wang resin, Fmoc-Tyr(tBu)-Wangresin, Fmoc-Val-Wang resin).

The term “stable moiety,” as used herein, preferably refers to a moietywhich possess stability sufficient to allow manufacture, and whichmaintains its integrity for a sufficient period of time to be useful forthe purposes detailed herein.

A “suitable amino-protecting group,” as used herein, is well known inthe art and include those described in detail in Protecting Groups inOrganic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, JohnWiley & Sons, 1999, the entirety of which is incorporated herein byreference. Suitable amino-protecting groups include methyl carbamate,ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative,N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonylderivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate,formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copperchelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), P-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

A “suitable carboxylic acid protecting group,” or “protected carboxylicacid,” as used herein, are well known in the art and include thosedescribed in detail in Greene (1999). Examples of suitably protectedcarboxylic acids further include, but are not limited to, silyl-,alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids.Examples of suitable silyl groups include trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and thelike. Examples of suitable alkyl groups include methyl, benzyl,p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl,tetrahydropyran-2-yl. Examples of suitable alkenyl groups include allyl.Examples of suitable aryl groups include optionally substituted phenyl,biphenyl, or naphthyl. Examples of suitable arylalkyl groups includeoptionally substituted benzyl (e.g., p-methoxybenzyl (MPM),3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl,2,6-dichlorobenzyl, p-cyanobenzyl), and 2- and 4-picolyl.

A “suitable hydroxyl protecting group” as used herein, is well known inthe art and include those described in detail in Protecting Groups inOrganic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, JohnWiley & Sons, 1999, the entirety of which is incorporated herein byreference. Suitable hydroxyl protecting groups include methyl,methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, co-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts). For protecting 1,2- or 1,3-diols, the protecting groups includemethylene acetal, ethylidene acetal, 1-t-butylethylidene ketal,1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal,2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal,cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal,p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal,3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal,methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethyleneortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine orthoester, 1,2-dimethoxyethylidene ortho ester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivative,α-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylideneortho ester, di-t-butylsilylene group (DTBS),1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cycliccarbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

A “suitable thiol protecting group,” as used herein, are well known inthe art and include those described in detail in Protecting Groups inOrganic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, JohnWiley & Sons, 1999, the entirety of which is incorporated herein byreference. Examples of suitably protected thiol groups further include,but are not limited to, thioesters, carbonates, sulfonates allylthioethers, thioethers, silyl thioethers, alkyl thioethers, arylalkylthioethers, and alkyloxyalkyl thioethers. Examples of suitable estergroups include formates, acetates, proprionates, pentanoates,crotonates, and benzoates. Specific examples of suitable ester groupsinclude formate, benzoyl formate, chloroacetate, trifluoroacetate,methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate,pivaloate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate,p-benylbenzoate, 2,4,6-trimethylbenzoate. Examples of suitablecarbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl,2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, andp-nitrobenzyl carbonate. Examples of suitable silyl groups includetrimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triisopropylsilyl ether, and other trialkylsilylethers. Examples of suitable alkyl groups include methyl, benzyl,p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allyl ether,or derivatives thereof. Examples of suitable arylalkyl groups includebenzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and4-picolyl ethers.

The term “thio,” or “thiol,” as used herein, refers to a group of theformula (—SH). A “substituted thiol” refers to a group of the formula(—SR^(r)), wherein R^(r) can be any substituten that results in theformation of a stable moiety (e.g., a suitable thiol protecting group;aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, sulfinyl, sulfonyl, cyano, nitro, alkylaryl,arylalkyl, and the like, each of which may or may not be furthersubstituted).

The term “thiooxo,” as used herein, refers to a group of the formula(═S).

As used herein, a “pharmaceutically acceptable form thereof” includesany pharmaceutically acceptable salts, prodrugs, tautomers, isomers,and/or polymorphs of a compound of the present invention, as definedbelow and herein.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid ormalonic acid or by using other methods used in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium, quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and arylsulfonate.

As used herein, the term “prodrug” refers to a derivative of a parentcompound that requires transformation within the body in order torelease the parent compound. In certain cases, a prodrug has improvedphysical and/or delivery properties over the parent compound. Prodrugsare typically designed to enhance pharmaceutically and/orpharmacokinetically based properties associated with the parentcompound. The advantage of a prodrug can lie in its physical properties,such as enhanced water solubility for parenteral administration atphysiological pH compared to the parent compound, or it enhancesabsorption from the digestive tract, or it may enhance drug stabilityfor long-term storage. In recent years several types of bioreversiblederivatives have been exploited for utilization in designing prodrugs.Using esters as a prodrug type for compounds containing a carboxyl orhydroxyl functionality is known in the art as described, for example, in“The Organic Chemistry of Drug Design and Drug Interaction” RichardSilverman, published by Academic Press (1992).

As used herein, the term “tautomer” includes two or moreinterconvertible compounds resulting from at least one formal migrationof a hydrogen atom and at least one change in valency (e.g., a singlebond to a double bond, a triple bond to a double bond, or vice versa).The exact ratio of the tautomers depends on several factors, includingtemperature, solvent, and pH. Tautomerizations (i.e., the reactionproviding a tautomeric pair) may catalyzed by acid or base. Exemplarytautomerizations include keto-to-enol; amide-to-imide; lactam-to-lactim;enamine-to-imine; and enamine-to-(a different) enamine tautomerizations.

As used herein, the term “isomers” includes any and all geometricisomers and stereoisomers. For example, “isomers” include cis- andtrans-isomers, E- and Z-isomers, R- and S-enantiomers, diastereomers,(D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixturesthereof, as falling within the scope of the invention. For instance, anisomer/enantiomer may, in some embodiments, be provided substantiallyfree of the corresponding enantiomer, and may also be referred to as“optically enriched.” “Optically-enriched,” as used herein, means thatthe compound is made up of a significantly greater proportion of oneenantiomer. In certain embodiments the compound of the present inventionis made up of at least about 90% by weight of a preferred enantiomer. Inother embodiments the compound is made up of at least about 95%, 98%, or99% by weight of a preferred enantiomer. Preferred enantiomers may beisolated from racemic mixtures by any method known to those skilled inthe art, including chiral high pressure liquid chromatography (HPLC) andthe formation and crystallization of chiral salts or prepared byasymmetric syntheses. See, for example, Jacques, et al., Enantiomers,Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen,S. H., et al., Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistryof Carbon Compounds (McGraw-Hill, N Y, 1962); Wilen, S. H. Tables ofResolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, Ind. 1972).

As used herein, “polymorph” refers to a crystalline inventive compoundexisting in more than one crystalline form/structure. When polymorphismexists as a result of difference in crystal packing it is called packingpolymorphism. Polymorphism can also result from the existence ofdifferent conformers of the same molecule in conformationalpolymorphism. In pseudopolymorphism the different crystal types are theresult of hydration or solvation.

The term “amino acid” refers to a molecule containing both an aminogroup and a carboxyl group. Amino acids include alpha-amino acids andbeta-amino acids, the structures of which are depicted below. In certainembodiments, an amino acid is an alpha amino acid.

Suitable amino acids include, without limitation, natural alpha-aminoacids such as D- and L-isomers of the 20 common naturally occurringalpha-amino acids found in peptides (e.g., A, R, N, C, D, Q, E, G, H, I,L, K, M, F, P, S, T, W, Y, V, as provided in Table 1 depicted below),unnatural alpha-amino acids (as depicted in Tables 2 and 3 below),natural beta-amino acids (e.g., beta-alanine), and unnatural beta-aminoacids.

Amino acids used in the construction of peptides of the presentinvention may be prepared by organic synthesis, or obtained by otherroutes, such as, for example, degradation of or isolation from a naturalsource. In certain embodiments of the present invention, the formula—[X_(AA)]— corresponds to the natural and/or unnatural amino acidshaving the following formulae:

wherein R and R′ correspond a suitable amino acid side chain, as definedbelow and herein, and R^(a) is as defined below and herein.

TABLE 1 Exemplary natural Suitable amino acid side chains alpha-aminoacids R R′ L-Alanine (A) —CH₃ —H L-Arginine (R) —CH₂CH₂CH₂—NHC(═NH)NH₂—H L-Asparagine (N) —CH₂C(═O)NH₂ —H L-Aspartic acid (D) —CH₂CO₂H —HL-Cysteine (C) —CH₂SH —H L-Glutamic acid (E) —CH₂CH₂CO₂H —H L-Glutamine(Q) —CH₂CH₂C(═O)NH₂ —H Glycine (G) —H —H L-Histidine (H)—CH₂-2-(1H-imidazole) —H L-Isoleucine (I) -sec-butyl —H L-Leucine (L)-iso-butyl —H L-Lysine (K) —CH₂CH₂CH₂CH₂NH₂ —H L-Methionine (M)—CH₂CH₂SCH₃ —H L-Phenylalanine (F) —CH₂Ph —H L-Proline (P)-2-(pyrrolidine) —H L-Serine (S) —CH₂OH —H L-Threonine (T)—CH₂CH(OH)(CH₃) —H L-Tryptophan (W) —CH₂-3-(1H-indole) —H L-Tyro sine(Y) —CH₂-(p-hydroxyphenyl) —H L-Valine (V) -isopropyl —H

TABLE 2 Exemplary unnatural Suitable amino acid side chains alpha-aminoacids R R′ D-Alanine —H —CH₃ D-Arginine —H —CH₂CH₂CH₂—NHC(═NH)NH₂D-Asparagine —H —CH₂C(═O)NH₂ D-Aspartic acid —H —CH₂CO₂H D-Cysteine —H—CH₂SH D-Glutamic acid —H —CH₂CH₂CO₂H D-Glutamine —H —CH₂CH₂C(═O)NH₂D-Histidine —H —CH₂-2-(1H-imidazole) D-Isoleucine —H -sec-butylD-Leucine —H -iso-butyl D-Lysine —H —CH₂CH₂CH₂CH₂NH₂ D-Methionine —H—CH₂CH₂SCH₃ D-Phenylalanine —H —CH₂Ph D-Proline —H -2-(pyrrolidine)D-Serine —H —CH₂OH D-Threonine —H —CH₂CH(OH)(CH₃) D-Tryptophan —H—CH₂-3-(1H-indole) D-Tyrosine —H —CH₂-(p-hydroxyphenyl) D-Valine —H-isopropyl Di-vinyl —CH═CH₂ —CH═CH₂ Exemplary unnatural alpha-aminoacids R and R′ are equal to: α-methyl-Alanine (Aib) —CH₃ —CH₃α-methyl-Arginine —CH₃ —CH₂CH₂CH₂—NHC(═NH)NH₂ α-methyl-Asparagine —CH₃—CH₂C(═O)NH₂ α-methyl-Aspartic acid —CH₃ —CH₂CO₂H α-methyl-Cysteine —CH₃—CH₂SH α-methyl-Glutamic acid —CH₃ —CH₂CH₂CO₂H α-methyl-Glutamine —CH₃—CH₂CH₂C(═O)NH₂ α-methyl-Histidine —CH₃ —CH₂-2-(1H-imidazole)α-methyl-Isoleucine —CH₃ -sec-butyl α-methyl-Leucine —CH₃ -iso-butylα-methyl-Lysine —CH₃ —CH₂CH₂CH₂CH₂NH₂ α-methyl-Methionine —CH₃—CH₂CH₂SCH₃ α-methyl-Phenylalanine —CH₃ —CH₂Ph α-methyl-Proline —CH₃-2-(pyrrolidine) α-methyl-Serine —CH₃ —CH₂OH α-methyl-Threonine —CH₃—CH₂CH(OH)(CH₃) α-methyl-Tryptophan —CH₃ —CH₂-3-(1H-indole)α-methyl-Tyrosine —CH₃ —CH₂-(p-hydroxyphenyl) α-methyl-Valine —CH₃-isopropyl Di-vinyl —CH═CH₂ —CH═CH₂ Norleucine —H —CH₂CH₂CH₂CH₃

TABLE 3 Exemplary unnatural alpha-amino acids Terminally unsaturatedalpha-amino acids and bis alpha-amino acids (e.g., modified cysteine,modified lysine, modified tryptophan, modified serine, modifiedthreonine, modified proline, modified histidine, modified alanine, andthe like). Suitable amino acid side chains R and R′ is equal to hydrogenof —CH₃ and: —(CH₂)_(g)—S—(CH₂)_(g)—CH═CH₂,—(CH₂)_(g)—O—(CH₂)_(g)—CH═CH₂, —(CH₂)_(g)—NH—(CH₂)_(g)—CH═CH₂,—(CH₂)_(g)—(C═O)—S—(CH₂)_(g)—CH═CH₂,—(CH₂)_(g)—(C═O)—O—(CH₂)_(g)—CH═CH₂,—(CH₂)_(g)—(C═O)—NH—(CH₂)_(g)—CH═CH₂, —CH₂CH₂CH₂CH₂—NH—(CH₂)_(g)CH═CH₂,—(C₆H₅)-p—O—(CH₂)_(g)CH═CH₂, —CH(CH₃)—O—(CH₂)_(g)CH═CH₂,—CH₂CH(—O—CH═CH₂)(CH₃), -histidine-N((CH₂)_(g)CH═CH₂),-tryptophan-N((CH₂)_(g)CH═CH₂), and —(CH₂)_(g+1)(CH═CH₂), wherein: eachinstance of g is, independently, 0 to 10. Exemplary unnaturalalpha-amino acids

R₅

R₈

S₅

S₈

B₅

There are many known unnatural amino acids any of which may be includedin the peptides of the present invention. See for example, S. Hunt, TheNon-Protein Amino Acids: In Chemistry and Biochemistry of the AminoAcids, edited by G. C. Barrett, Chapman and Hall, 1985. Some examples ofunnatural amino acids are 4-hydroxyproline, desmosine,gamma-aminobutyric acid, beta-cyanoalanine, norvaline,4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine,1-amino-cyclopropanecarboxylic acid,1-amino-2-phenyl-cyclopropanecarboxylic acid,1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic acid,3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid,4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid,2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminoheptanedioicacid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-, meta-and para-substituted phenylalanines (e.g., substituted with —C(═O)C₆H₅;—CF₃; —CN; -halo; —NO₂; CH₃), disubstituted phenylalanines, substitutedtyrosines (e.g., further substituted with —C(═O)C₆H₅; —CF₃; —CN; -halo;—NO₂; CH₃), and statine. Additionally, the amino acids suitable for usein the present invention may be derivatized to include amino acidresidues that are hydroxylated, phosphorylated, sulfonated, acylated,and glycosylated, to name a few.

The term “amino acid side chain” refers to a group attached to thealpha- or beta-carbon of an amino acid. A “suitable amino acid sidechain” includes, but is not limited to, any of the suitable amino acidside chains as defined above, and as provided in Tables 1 to 3.

For example, suitable amino acid side chains include methyl (as thealpha-amino acid side chain for alanine is methyl),4-hydroxyphenylmethyl (as the alpha-amino acid side chain for tyrosineis 4-hydroxyphenylmethyl) and thiomethyl (as the alpha-amino acid sidechain for cysteine is thiomethyl), etc. A “terminally unsaturated aminoacid side chain” refers to an amino acid side chain bearing a terminalunsaturated moiety, such as a substituted or unsubstituted, double bond(e.g., olefinic) or a triple bond (e.g., acetylenic), that participatesin crosslinking reaction with other terminal unsaturated moieties in thepolypeptide chain. In certain embodiments, a “terminally unsaturatedamino acid side chain” is a terminal olefinic amino acid side chain. Incertain embodiments, a “terminally unsaturated amino acid side chain” isa terminal acetylenic amino acid side chain. In certain embodiments, theterminal moiety of a “terminally unsaturated amino acid side chain” isnot further substituted. Terminally unsaturated amino acid side chainsinclude, but are not limited to, side chains as depicted in Table 3.

A “peptide” or “polypeptide” comprises a polymer of amino acid residueslinked together by peptide (amide) bonds. The term(s), as used herein,refers to proteins, polypeptides, and peptide of any size, structure, orfunction. Typically, a peptide or polypeptide will be at least threeamino acids long. A peptide or polypeptide may refer to an individualprotein or a collection of proteins. Inventive proteins preferablycontain only natural amino acids, although non-natural amino acids(i.e., compounds that do not occur in nature but that can beincorporated into a polypeptide chain) and/or amino acid analogs as areknown in the art may alternatively be employed. Also, one or more of theamino acids in a peptide or polypeptide may be modified, for example, bythe addition of a chemical entity such as a carbohydrate group, ahydroxyl group, a phosphate group, a farnesyl group, an isofarnesylgroup, a fatty acid group, a linker for conjugation, functionalization,or other modification, etc. A peptide or polypeptide may also be asingle molecule or may be a multi-molecular complex, such as a protein.A peptide or polypeptide may be just a fragment of a naturally occurringprotein or peptide. A peptide or polypeptide may be naturally occurring,recombinant, or synthetic, or any combination thereof. As used herein“dipeptide” refers to two covalently linked amino acids.

The Following Definitions are More General Terms Used Throughout thePresent Application:

The term “subject,” as used herein, refers to any animal. In certainembodiments, the subject is a mammal. In certain embodiments, the term“subject”, as used herein, refers to a human (e.g., a man, a woman, or achild).

The terms “administer,” “administering,” or “administration,” as usedherein refers to implanting, absorbing, ingesting, injecting, orinhaling, the inventive polypeptide or compound.

The terms “treat” or “treating,” as used herein, refers to partially orcompletely alleviating, inhibiting, ameliorating, and/or relieving thedisease or condition from which the subject is suffering.

The terms “effective amount” and “therapeutically effective amount,” asused herein, refer to the amount or concentration of a biologicallyactive agent conjugated to an inventive polypeptide of the presentlyclaimed invention, or amount or concentration of an inventivepolypeptide, that, when administered to a subject, is effective to atleast partially treat a condition from which the subject is suffering.

As used herein, when two entities are “conjugated” to one another theyare linked by a direct or indirect covalent or non-covalent interaction.In certain embodiments, the association is covalent. In otherembodiments, the association is non-covalent. Non-covalent interactionsinclude hydrogen bonding, van der Waals interactions, hydrophobicinteractions, magnetic interactions, electrostatic interactions, etc. Anindirect covalent interaction is when two entities are covalentlyconnected, optionally through a linker group.

As used herein, a “biologically active agent” or “therapeutically activeagent” refers to any substance used as a medicine for treatment,prevention, delay, reduction or amelioration of a disease, condition, ordisorder, and refers to a substance that is useful for therapy,including prophylactic and therapeutic treatment. A biologically activeagent also includes a compound that increases the effect oreffectiveness of another compound, for example, by enhancing potency orreducing adverse effects of the other compound.

In certain embodiments, a biologically active agent is an anti-canceragent, antibiotic, anti-viral agent, anti-HIV agent, anti-parasiteagent, anti-protozoal agent, anesthetic, anticoagulant, inhibitor of anenzyme, steroidal agent, steroidal or non-steroidal anti-inflammatoryagent, antihistamine, immunosuppressant agent, anti-neoplastic agent,antigen, vaccine, antibody, decongestant, sedative, opioid, analgesic,anti-pyretic, birth control agent, hormone, prostaglandin,progestational agent, anti-glaucoma agent, ophthalmic agent,anti-cholinergic, analgesic, anti-depressant, anti-psychotic,neurotoxin, hypnotic, tranquilizer, anti-convulsant, muscle relaxant,anti-Parkinson agent, anti-spasmodic, muscle contractant, channelblocker, miotic agent, anti-secretory agent, anti-thrombotic agent,anticoagulant, anti-cholinergic, β-adrenergic blocking agent, diuretic,cardiovascular active agent, vasoactive agent, vasodilating agent,anti-hypertensive agent, angiogenic agent, modulators ofcell-extracellular matrix interactions (e.g. cell growth inhibitors andanti-adhesion molecules), or inhibitors/intercalators of DNA, RNA,protein-protein interactions, protein-receptor interactions, etc.

Exemplary biologically active agents include, but are not limited to,small organic molecules such as drug compounds, peptides, proteins,carbohydrates, monosaccharides, oligosaccharides, polysaccharides,nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides orproteins, small molecules linked to proteins, glycoproteins, steroids,nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides,antisense oligonucleotides, lipids, hormones, vitamins, and cells. Incertain embodiments, the biologically active agent is a cell. Exemplarycells include immune system cells (e.g., mast, lymphocyte, plasma cell,macrophage, dendritic cell, neutrophils, eosinophils), connective tissuecells (e.g., blood cells, erythrocytes, leucocytes, megakarocytes,fibroblasts, osteoclasts), stem cells (e.g., embryonic stem cells, adultstem cells), bone cells, glial cells, pancreatic cells, kidney cells,nerve cells, skin cells, liver cells, muscle cells, adipocytes, Schwanncells, Langerhans cells, as well as (micro)-tissues such as the Isletsof Langerhans.

In certain embodiments, the biologically active agent is a small organicmolecule. In certain embodiments, a small organic molecule isnon-peptidic. In certain embodiments, a small organic molecule isnon-oligomeric. In certain embodiments, a small organic molecule is anatural product or a natural product-like compound having a partialstructure (e.g., a substructure) based on the full structure of anatural product. Exemplary natural products include steroids,penicillins, prostaglandins, venoms, toxins, morphine, paclitaxel(Taxol), morphine, cocaine, digitalis, quinine, tubocurarine, nicotine,muscarine, artemisinin, cephalosporins, tetracyclines, aminoglycosides,rifamycins, chloramphenicol, asperlicin, lovastatin, ciclosporin,curacin A, eleutherobin, discodermolide, bryostatins, dolostatins,cephalostatins, antibiotic peptides, epibatidine, α-bungarotoxin,tetrodotoxin, teprotide, and neurotoxins from Clostridium botulinum. Incertain embodiments, a small organic molecule is a drug approved by theFood and Drugs Administration as provided in the Code of FederalRegulations (CFR).

As used herein, a “label” refers to a moiety that has at least oneelement, isotope, or functional group incorporated into the moiety whichenables detection of the inventive polypeptide to which the label isattached. Labels can be directly attached (ie, via a bond) or can beattached by a linker (e.g., such as, for example, a cyclic or acyclic,branched or unbranched, substituted or unsubstituted alkylene; cyclic oracyclic, branched or unbranched, substituted or unsubstitutedalkenylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted alkynylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroalkylene; cyclic or acyclic, branchedor unbranched, substituted or unsubstituted heteroalkenylene; cyclic oracyclic, branched or unbranched, substituted or unsubstitutedheteroalkynylene; substituted or unsubstituted arylene; substituted orunsubstituted heteroarylene; or substituted or unsubstituted acylene, orany combination thereof, which can make up a linker). It will beappreciated that the label may be attached to the inventive polypeptideat any position that does not interfere with the biological activity orcharacteristic of the inventive polypeptide that is being detected.

In general, a label can fall into any one (or more) of five classes: a)a label which contains isotopic moieties, which may be radioactive orheavy isotopes, including, but not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N,³¹P, ³²P, ³⁵S, ⁶⁷Ga, ^(99m)Tc (Tc-⁹⁹m), ¹¹¹In, ¹²³I, ¹²⁵I, ¹⁶⁹Yb, and¹⁸⁶Re; b) a label which contains an immune moiety, which may beantibodies or antigens, which may be bound to enzymes (e.g., such ashorseradish peroxidase); c) a label which is a colored, luminescent,phosphorescent, or fluorescent moieties (e.g., such as the fluorescentlabel FITC); d) a label which has one or more photoaffinity moieties;and e) a label which has a ligand moiety with one or more known bindingpartners (such as biotin-streptavidin, FK506-FKBP, etc.). Any of thesetype of labels as described above may also be referred to as “diagnosticagents” as defined herein.

In certain embodiments, such as in the identification of a biologicaltarget, label comprises a radioactive isotope, preferably an isotopewhich emits detectable particles, such as β particles. In certainembodiments, the label comprises one or more photoaffinity moieties forthe direct elucidation of intermolecular interactions in biologicalsystems. A variety of known photophores can be employed, most relying onphotoconversion of diazo compounds, azides, or diazirines to nitrenes orcarbenes (see, Bayley, H., Photogenerated Reagents in Biochemistry andMolecular Biology (1983), Elsevier, Amsterdam, the entire contents ofwhich are incorporated herein by reference). In certain embodiments ofthe invention, the photoaffinity labels employed are o-, m- andp-azidobenzoyls, substituted with one or more halogen moieties,including, but not limited to 4-azido-2,3,5,6-tetrafluorobenzoic acid.

In certain embodiments, the label comprises one or more fluorescentmoieties. In certain embodiments, the label is the fluorescent labelFITC. In certain embodiments, the label comprises a ligand moiety withone or more known binding partners. In certain embodiments, the labelcomprises the ligand moiety biotin.

As used herein, a “diagnostic agent” refers to imaging agents. Exemplaryimaging agents include, but are not limited to, those used in positronemissions tomography (PET), computer assisted tomography (CAT), singlephoton emission computerized tomography, x-ray, fluoroscopy, andmagnetic resonance imaging (MRI); anti-emetics; and contrast agents.Exemplary diagnostic agents include but are not limited to, fluorescentmoieties, luminescent moieties, magnetic moieties; gadolinium chelates(e.g., gadolinium chelates with DTPA, DTPA-BMA, DOTA and HP-DO3A), ironchelates, magnesium chelates, manganese chelates, copper chelates,chromium chelates, iodine-based materials useful for CAT and x-rayimaging, and radionuclides. Suitable radionuclides include, but are notlimited to, ¹²³I, ¹²⁵I, ¹³⁰I, ¹³¹I, ¹³³I, ¹³⁵I, ⁴⁷Sc, ⁷²As, ⁷²Se, ⁹⁰Y,⁸⁸Y, ⁹⁷Ru, ¹⁰⁰Pd, ¹⁰¹mRh, ¹¹⁹Sb, ¹²⁸Ba, ¹⁹⁷Hg, ²¹¹At, ²¹²Bi, ²¹²Pb,¹⁰⁹Pd, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ⁶⁷Cu, ⁷⁵Br, ⁷⁷Br, ^(99m)Tc, ¹⁴C, ¹³N, ¹⁵O,³²P, ³³P, and ¹⁸F. Fluorescent and luminescent moieties include, but arenot limited to, a variety of different organic or inorganic smallmolecules commonly referred to as “dyes,” “labels,” or “indicators.”Examples include, but are not limited to, fluorescein, rhodamine,acridine dyes, Alexa dyes, cyanine dyes, etc. Fluorescent andluminescent moieties may include a variety of naturally occurringproteins and derivatives thereof, e.g., genetically engineered variants.For example, fluorescent proteins include green fluorescent protein(GFP), enhanced GFP, red, blue, yellow, cyan, and sapphire fluorescentproteins, reef coral fluorescent protein, etc. Luminescent proteinsinclude luciferase, aequorin and derivatives thereof. Numerousfluorescent and luminescent dyes and proteins are known in the art (see,e.g., U.S. Patent Publication 2004/0067503; Valeur, B., “MolecularFluorescence: Principles and Applications,” John Wiley and Sons, 2002;and Handbook of Fluorescent Probes and Research Products, MolecularProbes, 9^(th) edition, 2002).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Synthesis of stitched α-helical peptides by tandem ring-closingolefin metathesis. (A) Schematic structure of a α-helical tetra-olefinicpeptide designed to undergo tandem-RCM. Three regioisomeric tandem-RCMpathways are possible (a+b, c+d, and e+f); these would yield products 2,3, and 4, respectively. (B) Schematic structure of the sole product, thestitched peptide 4. The stereochemical configuration of the spiro carbon(red dot) and the N-terminal olefin were established by modeling; thatof the C-terminal olefin was not unambiguously established but isexpected to be trans. (C) Schematic structure of the product of an i+4+4crosslinking reaction, the stitched peptide 8. The stereochemicalconfiguration of the spiro carbon (red dot) and the olefins wereestablished by modeling. (D) Olefin-bearing amino acids used in thisstudy. (A-D) Blue groups face forward in these views; red backward.

FIGS. 2A-2C. Temperature-dependent circular dichroism spectra of (A) 5,and (B) 4. Inset: thermal melting curves and T_(m). (C) Comparison ofthe rates of trypsin digestion of 4 versus 5.

FIGS. 3A-3C. Temperature-dependent circular dichroism spectra of (A)peptide 9 (97 μM), (B) 6 (98 μM), (C) 8 (94 μM).

FIG. 4. Thermal melting curves and T_(m).

FIG. 5. HPLC chromatogram of purified peptide 9. 10-64% B for 0-12 min;64-10% B for 1215 min; 10% B for 15-18 min on an Agilent C₁₈ reversephase column (3.5×150 mm); A: 0.1% TFA in H₂O, B: acetonitrile; flowrate: 0.5 mL/min.

FIG. 6. HPLC chromatogram of purified peptide 4. 50-85% B for 0-14 min;85-50% for 14-18 min on an Agilent C₁₈ reverse phase column (3.5×150mm); A: 0.1% TFA in H₂O, B: acetonitrile; flow rate: 0.5 mL/min.

FIG. 7. HPLC chromatogram of purified peptide 6. 10-100% B for 0-20 min;100% B for 20-25 min; 100-10% B for 25-30 min 10% B for 30-35 min on anAgilent C18 reverse phase column (3.5×150 mm); A: 0.1% TFA in H₂O, B:acetonitrile; flow rate: 0.5 mL/min.

FIG. 8. HPLC chromatogram of purified peptide 5. 10-100% B for 0-20 min;100% B for 20-25 min; 100-10% B for 25-30 min 10% B for 30-35 min on anAgilent C18 reverse phase column (3.5×150 mm); A: 0.1% TFA in H₂O, B:acetonitrile; flow rate: 0.5 mL/min.

FIG. 9. HPLC chromatogram of purified peptide 8. 50-85% B for 0-14 min;85-50% for 14-18 min on an Agilent C₁₈ reverse phase column (3.5×150mm); A: 0.1% TFA in H₂O, B: acetonitrile; flow rate: 0.5 mL/min.

FIG. 10. Schematic structures of peptides 3, 4, 8, and 16.

FIG. 11. Graphical representation of the global minimum peptide 4 (A andB) and peptide 3 (C and D). The N-termini lie on the bottom ends of thepeptides. Views B and D depict ˜90° rotations of A and C, respectively.The alpha-carbons attached to the staple are depicted as spheres, whilethe olefin moiety is colored red.

FIG. 12. Graphical representation of the global minimum peptide 8 (A andB) and peptide 16 (C and D) stitched peptides. The N-termini lie on thebottom ends of the peptides. Views B and D depict ˜90° rotations of Aand C, respectively. The α-carbons attached to the staple are depictedas spheres, while the olefin moiety is colored red.

FIG. 13. Triple stitching via tandem ring-closing metathesis ofpolyalanine-based peptide(S5-Ala-Ala-Ala-B5-Ala-Ala-Ala-B5-Ala-Ala-Ala-S5) on resin.

FIG. 14. HPLC chromatogram at 0, 5, 10, 20, 30, 60, 90, 120, 165 minutesof ring-closing metathesis of of polyalanine-based peptide using 30%Grubbs catalyst

FIG. 15. A model peptide bearing B₅ at i and i+4 (peptide 25) did notproduce double stitched compound 27, and provided only singly stapledproduct 26. In addition, a model peptide containing R₅ at i and S₅ ati+4 position (peptide 28) did not undergo RCM. The results from thismodel study indicated that peptide 24 of FIG. 13 to be the most likelystructure for the triply stitched product. This result suggest that fouror more crosslinks also might be introduced to peptide system byrational design.

FIG. 16. The alpha-helix of BID BH3 domain (SAHBa) as reported inWalensky et al. Science (2004) 305:1466, was stabilized by stapling, asreported herein, and subjected to the cytochrome C release assay, asreported therein. One of the tandem RCM products, peptide 34, which isshorter than SAHBa by 8 residues, showed similar potency in cytochrome Creleasing effect, likely via a pro-apoptotic BAX/BAK pathway. Thepeptide 34 showed lower binding affinity for anti-apoptotic proteinBCL-XL, suggesting this peptide might have higher specificity for BAXprotein than SAHBa does.

FIG. 17. Depiction of the synthesis of alpha-methyl-alpha-terminallyunsaturated amino acids as described by U.S. Patent ApplicationPublication No. 2005/0250680.

FIG. 18. Depiction of the synthesis of alpha-methyl-alpha-terminallyunsaturated amino acids as described by U.S. Patent ApplicationPublication No. 2006/0008848.

FIG. 19. Exemplary reaction mechanism for a ring closing metathesis(RCM) reaction using a ruthenium (Grubbs) catalyst.

FIG. 20. Uptake of stitched peptides by Jurkat cells in a quantitativeimmunofluorescence assay. Stitched (“multiply stapled”) peptides showcompatible cell permeability compared to their singly “stapled” analogs.

FIGS. 21A-21D. Stabilities of peptides against guanidine hydrochloride.Stitched peptide 4 displays a high level of stability against thedenaturing agent as it remains fully helical even at extremely highconcentrations of guanidine salt.

FIGS. 22A-22B. Stabilities of peptides against proteases. Stitchedpeptide 4 shows a higher level of stability against both trypsin (A) andchymotrypsin (B) than stapled peptide 5.

FIGS. 23A-23F. Circular dichroism spectra of stitched peptides withvarious constitutions. Triple stitched peptide Id shows a high level ofthermal stability.

FIGS. 24A-24C. Cell permeabilities of FITC-labeled peptides analyzed byFACS at 37° C.

FIGS. 25A-25C. Temperature-dependent cell penetration of peptides.Stitched peptide IIe is less affected by low temperature compared tostapled peptide IId.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

The present invention provides novel polypeptides comprising (i) atleast two amino acids, each comprising at least one terminallyunsaturated amino acid sidechain, and (ii) at least one amino acidcomprising at least two terminally unsaturated amino acid side chains.Such polypeptides may be reacted under suitable conditions to forminventive stabilized “stitched” polypeptides. In certain embodiments,these multiple “staples,” or cross-links, which comprise the “stitch”are used to stabilize the polypeptides secondary structure (e.g., analpha helix).

The present invention also provides pharmaceutical compositionscomprising an inventive stitched polypeptide. Furthermore, the presentinvention provides methods of making and using inventive stitchedpolypeptides.

Inventive stitched polypeptides, as described herein, may be usefulwherever such stabilized secondary structural motifs are advantageous,for example, as a therapeutic agent, as a biological probe, or as a drugdelivery agent. The inventive peptides may function as modulators ofprotein-protein, protein-ligand, or protein-receptor bindinginteractions. In certain embodiments, these inventive stitchedpolypeptides are useful in the treatment of proliferative, neurological,immunological, endocrinologic, cardiovascular, hematologic, and/orinflammatory diseases, disorders, and/or conditions, and conditionscharacterized by premature or unwanted cell death.

Exemplary secondary structural motifs of polypeptides and proteinsinclude, but are not limited to, an alpha-helix, alpha-L, 3₁₀ helix, πhelix, and type II helices (e.g., left-handed helices). In certainembodiments, the predominant secondary structural motif of the inventivepolypeptide is an alpha helix.

In one aspect, the present invention provides an “unstitched”polypeptide of the formula (I):

wherein:

each instance of K, L₁, L₂, and M, is, independently, a bond, cyclic oracyclic, branched or unbranched, substituted or unsubstituted alkylene;cyclic or acyclic, branched or unbranched, substituted or unsubstitutedalkenylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted alkynylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroalkylene; cyclic or acyclic, branchedor unbranched, substituted or unsubstituted heteroalkenylene; cyclic oracyclic, branched or unbranched, substituted or unsubstitutedheteroalkynylene; substituted or unsubstituted arylene; substituted orunsubstituted heteroarylene; or substituted or unsubstituted acylene;

each instance of R^(a) is, independently, hydrogen; cyclic or acyclic,branched or unbranched, substituted or unsubstituted aliphatic; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; cyclic or acyclic, substituted orunsubstituted acyl; or R^(a) is a suitable amino protecting group;

each instance of R^(b) is, independently, a suitable amino acid sidechain; hydrogen; cyclic or acyclic, branched or unbranched, substitutedor unsubstituted aliphatic; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; cyclic oracyclic, substituted or unsubstituted acyl; substituted or unsubstitutedhydroxyl; substituted or unsubstituted thiol; substituted orunsubstituted amino; cyano; isocyano; halo; or nitro;

each instance of R^(c), is, independently, hydrogen; cyclic or acyclic,branched or unbranched, substituted or unsubstituted aliphatic; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; cyclic or acyclic, substituted orunsubstituted acyl; substituted or unsubstituted hydroxyl; substitutedor unsubstituted thiol; substituted or unsubstituted amino; cyano;isocyano; halo; or nitro;

each instance of R^(e) is, independently, —R^(E), —OR^(E), —N(R^(E))₂,or —SR^(E), wherein each instance of R^(E) is, independently, hydrogen,cyclic or acyclic, branched or unbranched, substituted or unsubstitutedaliphatic; cyclic or acyclic, branched or unbranched, substituted orunsubstituted heteroaliphatic; substituted or unsubstituted aryl;substituted or unsubstituted heteroaryl; substituted or unsubstitutedacyl; a resin; a suitable hydroxyl, amino, or thiol protecting group; ortwo R^(E) groups together form a substituted or unsubstituted 5- to6-membered heterocyclic or heteroaromatic ring;

each instance of R^(f) is, independently, hydrogen; cyclic or acyclic,branched or unbranched, substituted or unsubstituted aliphatic; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; substituted or unsubstituted acyl; a resin; asuitable amino protecting group; a label optionally joined by a linker,wherein the linker is selected from cyclic or acyclic, branched orunbranched, substituted or unsubstituted alkylene; cyclic or acyclic,branched or unbranched, substituted or unsubstituted alkenylene; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedalkynylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted heteroalkylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroalkenylene; cyclic or acyclic,branched or unbranched, substituted or unsubstituted heteroalkynylene;substituted or unsubstituted arylene; substituted or unsubstitutedheteroarylene; or substituted or unsubstituted acylene; or R^(f) andR^(a) together form a substituted or unsubstituted 5- to 6-memberedheterocyclic or heteroaromatic ring;

each instance of X_(A)a is, independently, a natural or unnatural aminoacid;

each instance of x is, independently, an integer between 0 to 3;

each instance of y and z is, independently, an integer between 2 to 6;

each instance of j is, independently, an integer between 1 to 10;

each instance of p is, independently, an integer between 0 to 10;

each instance of s and t is, independently, an integer between 0 and100;

each instance of u, v, and q, is, independently, an integer between 0 to4;

and wherein:

corresponds to a double or triple bond.

As is understood by one skilled in the art, R^(f) corresponds to theN-terminus and R^(e) corresponds to the C-terminus of the peptide chain.

Under suitable reaction conditions, a “stitched” polypeptide of theformulae (II) is generated from a polypeptide of formula (I):

wherein:

each instance of K, L₁, L₂, and M, is, independently, a bond, cyclic oracyclic, branched or unbranched, substituted or unsubstituted alkylene;cyclic or acyclic, branched or unbranched, substituted or unsubstitutedalkenylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted alkynylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroalkylene; cyclic or acyclic, branchedor unbranched, substituted or unsubstituted heteroalkenylene; cyclic oracyclic, branched or unbranched, substituted or unsubstitutedheteroalkynylene; substituted or unsubstituted arylene; substituted orunsubstituted heteroarylene; or substituted or unsubstituted acylene;

each instance of R^(a) is, independently, hydrogen; cyclic or acyclic,branched or unbranched, substituted or unsubstituted aliphatic; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; cyclic or acyclic, substituted orunsubstituted acyl; or R^(a) is a suitable amino protecting group;

each instance of R^(b) is, independently, a suitable amino acid sidechain; hydrogen; cyclic or acyclic, branched or unbranched, substitutedor unsubstituted aliphatic; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; cyclic oracyclic, substituted or unsubstituted acyl; substituted or unsubstitutedhydroxyl; substituted or unsubstituted thiol; substituted orunsubstituted amino; cyano; isocyano; halo; or nitro;

each instance of R^(e), is, independently, hydrogen; cyclic or acyclic,branched or unbranched, substituted or unsubstituted aliphatic; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; cyclic or acyclic, substituted orunsubstituted acyl; substituted or unsubstituted hydroxyl; substitutedor unsubstituted thiol; substituted or unsubstituted amino; cyano;isocyano; halo; or nitro;

each instance of R^(e) is, independently, —R^(E), —OR^(E), —N(R^(E))₂,or —SR^(E), wherein each instance of R^(E) is, independently, hydrogen,cyclic or acyclic, branched or unbranched, substituted or unsubstitutedaliphatic; cyclic or acyclic, branched or unbranched, substituted orunsubstituted heteroaliphatic; substituted or unsubstituted aryl;substituted or unsubstituted heteroaryl; substituted or unsubstitutedacyl; a resin; a suitable hydroxyl, amino, or thiol protecting group; ortwo R^(E) groups together form a substituted or unsubstituted 5- to6-membered heterocyclic or heteroaromatic ring;

each instance of R^(f) is, independently, hydrogen; cyclic or acyclic,branched or unbranched, substituted or unsubstituted aliphatic; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; substituted or unsubstituted acyl; a resin; asuitable amino protecting group; a label optionally joined by a linker,wherein the linker is selected from cyclic or acyclic, branched orunbranched, substituted or unsubstituted alkylene; cyclic or acyclic,branched or unbranched, substituted or unsubstituted alkenylene; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedalkynylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted heteroalkylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroalkenylene; cyclic or acyclic,branched or unbranched, substituted or unsubstituted heteroalkynylene;substituted or unsubstituted arylene; substituted or unsubstitutedheteroarylene; or substituted or unsubstituted acylene; substituted orunsubstituted acyl; or R^(f) and R^(a) together form a substituted orunsubstituted 5- to 6-membered heterocyclic or heteroaromatic ring;

each instance of R^(KL), R^(LL), and R^(LM), is, independently,hydrogen; cyclic or acyclic, branched or unbranched, substituted orunsubstituted aliphatic; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; substitutedor unsubstituted acyl; substituted or unsubstituted hydroxyl;substituted or unsubstituted thiol; substituted or unsubstituted amino;azido; cyano; isocyano; halo; nitro;

or two adjacent R^(KL) groups are joined to form a substituted orunsubstituted 5- to 8-membered cycloaliphatic ring; substituted orunsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted orunsubstituted aryl ring; or substituted or unsubstituted heteroarylring; two adjacent R^(KL) groups are joined to form a substituted orunsubstituted 5- to 8-membered cycloaliphatic ring; substituted orunsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted orunsubstituted aryl ring; or substituted or unsubstituted heteroarylring; or two adjacent R^(LM) groups are joined to form a substituted orunsubstituted 5- to 8-membered cycloaliphatic ring; substituted orunsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted orunsubstituted aryl ring; or substituted or unsubstituted heteroarylring;

each instance of X_(AA) is, independently, a natural or unnatural aminoacid;

each instance of x is, independently, an integer between 0 to 3;

each instance of y and z is, independently, an integer between 2 to 6;

each instance of j is, independently, an integer between 1 to 10;

each instance of p is, independently, an integer between 0 to 10;

each instance of s and t is, independently, an integer between 0 and100;

each instance of u, v, and q, is, independently, an integer between 0 to4;

and wherein:

corresponds to a double or triple bond; and

corresponds to a single, double, or triple bond.

As will be appreciated by one of skill in the art, a partially“stitched” polypeptide of the formulae (III) to (VII) may also begenerated from a polypeptide of formula (I) under suitable reactionconditions:

wherein:

each instance of K, L₁, L₂, and M, is, independently, a bond, cyclic oracyclic, branched or unbranched, substituted or unsubstituted alkylene;cyclic or acyclic, branched or unbranched, substituted or unsubstitutedalkenylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted alkynylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroalkylene; cyclic or acyclic, branchedor unbranched, substituted or unsubstituted heteroalkenylene; cyclic oracyclic, branched or unbranched, substituted or unsubstitutedheteroalkynylene; substituted or unsubstituted arylene; substituted orunsubstituted heteroarylene; or substituted or unsubstituted acylene;

each instance of R^(a) is, independently, hydrogen; cyclic or acyclic,branched or unbranched, substituted or unsubstituted aliphatic; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; cyclic or acyclic, substituted orunsubstituted acyl; or R^(a) is a suitable amino protecting group;

each instance of R^(b) is, independently, a suitable amino acid sidechain; hydrogen; cyclic or acyclic, branched or unbranched, substitutedor unsubstituted aliphatic; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; cyclic oracyclic, substituted or unsubstituted acyl; substituted or unsubstitutedhydroxyl; substituted or unsubstituted thiol; substituted orunsubstituted amino; cyano; isocyano; halo; or nitro;

each instance of R^(e), is, independently, hydrogen; cyclic or acyclic,branched or unbranched, substituted or unsubstituted aliphatic; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; cyclic or acyclic, substituted orunsubstituted acyl; substituted or unsubstituted hydroxyl; substitutedor unsubstituted thiol; substituted or unsubstituted amino; cyano;isocyano; halo; or nitro;

each instance of R^(e) is, independently, —R^(E), —OR^(E), —N(R^(E))₂,or —SR^(E), wherein each instance of R^(E) is, independently, hydrogen,cyclic or acyclic, branched or unbranched, substituted or unsubstitutedaliphatic; cyclic or acyclic, branched or unbranched, substituted orunsubstituted heteroaliphatic; substituted or unsubstituted aryl;substituted or unsubstituted heteroaryl; substituted or unsubstitutedacyl; a resin; a suitable hydroxyl, amino, or thiol protecting group; ortwo R^(E) groups together form a substituted or unsubstituted 5- to6-membered heterocyclic or heteroaromatic ring;

each instance of R^(r) is, independently, hydrogen; cyclic or acyclic,branched or unbranched, substituted or unsubstituted aliphatic; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; substituted or unsubstituted acyl; a resin; asuitable amino protecting group; a label optionally joined by a linker,wherein the linker is selected from cyclic or acyclic, branched orunbranched, substituted or unsubstituted alkylene; cyclic or acyclic,branched or unbranched, substituted or unsubstituted alkenylene; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedalkynylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted heteroalkylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroalkenylene; cyclic or acyclic,branched or unbranched, substituted or unsubstituted heteroalkynylene;substituted or unsubstituted arylene; substituted or unsubstitutedheteroarylene; or substituted or unsubstituted acylene; or R^(f) andR^(a) together form a substituted or unsubstituted 5- to 6-memberedheterocyclic or heteroaromatic ring;

each instance of R^(KL), R^(LL), and R^(LM), is, independently,hydrogen; cyclic or acyclic, branched or unbranched, substituted orunsubstituted aliphatic; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; substitutedor unsubstituted acyl; substituted or unsubstituted hydroxyl;substituted or unsubstituted thiol; substituted or unsubstituted amino;azido; cyano; isocyano; halo; nitro;

or two adjacent R^(KL) groups are joined to form a substituted orunsubstituted 5- to 8-membered cycloaliphatic ring; substituted orunsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted orunsubstituted aryl ring; or substituted or unsubstituted heteroarylring; two adjacent R^(KL) groups are joined to form a substituted orunsubstituted 5- to 8-membered cycloaliphatic ring; substituted orunsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted orunsubstituted aryl ring; or substituted or unsubstituted heteroarylring; or two adjacent R^(LM) groups are joined to form a substituted orunsubstituted 5- to 8-membered cycloaliphatic ring; substituted orunsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted orunsubstituted aryl ring; or substituted or unsubstituted heteroarylring;

each instance of X_(AA) is, independently, a natural or unnatural aminoacid;

each instance of x is, independently, an integer between 0 to 3;

each instance of y and z is, independently, an integer between 2 to 6;

each instance of j is, independently, an integer between 1 to 10;

each instance of p is, independently, an integer between 0 to 10;

each instance of s and t is, independently, an integer between 0 and100;

each instance of u, v, and q, is, independently, an integer between 0 to4;

and wherein:

corresponds to a double or triple bond; and

corresponds to a single, double, or triple bond.

In certain embodiments,

corresponds to a double bond.

In certain embodiments,

corresponds to a triple bond.

In certain embodiments,

corresponds to a single bond.

In certain embodiments,

corresponds to a double bond.

In certain embodiments,

corresponds to a triple bond.

In certain embodiments, the polypeptide of the above formulae (I), (II),(III), (IV), (V), (VI), or (VII) is an alpha-helical polypeptide. Incertain embodiments, the polypeptide of the above formulae (I), (II),(III), (IV), (V), (VI), or (VII) is a substantially alpha-helicalpolypeptide. As used herein, the phrase “substantially alpha-helical”refers to a polypeptide adopting, on average, backbone (φ, ψ) dihedralangles in a range from about (−90°, −15°) to about (−350, −700).Alternatively, the phrase “substantially alpha-helical” refers to apolypeptide adopting dihedral angles such that the ψ dihedral angle ofone residue and the φ dihedral angle of the next residue sums, onaverage, about −80° to about −125°. In certain embodiments, theinventive polypeptide adopts dihedral angles such that the ψ dihedralangle of one residue and the φ dihedral angle of the next residue sums,on average, about −100° to about −110°. In certain embodiments, theinventive polypeptide adopts dihedral angles such that the ψ dihedralangle of one residue and the φ dihedral angle of the next residue sums,on average, about −105°. Furthermore, the phrase “substantiallyalpha-helical” may also refer to a polypeptide having at least 50%, 60%,70%, 80%, 90%, or 95% of the amino acids provided in the polypeptidechain in an alpha-helical conformation, or with dihedral angles asspecified above and herein. Confirmation of a polypeptide'salpha-helical secondary structure may be ascertained by well-knownanalytical techniques, such as x-ray crystallography, electroncrystallography, fiber diffraction, fluorescence anisotropy, circulardichrosim (CD), and nuclear magnetic resonance spectroscopy.

In certain embodiments, the present invention provides a polypeptide ofthe formulae:

wherein K, M, L₁, L₂, R^(a), R^(b), R^(c), R^(e), R^(f), X_(AA), R^(KL),R^(LL), R^(LM), s, t, j, p, y, z, v, u, q, are as defined and describedabove and herein;wherein

corresponds to a single or double bond; andwherein u, v and q are, independently, 0, 1, 2, 3, or 4.

In certain embodiments, all

corresponds to a single bond, and u, v and q are, independently, 0, 1,2, 3, or 4.

In certain embodiments, all

corresponds to a double bond, u, v and q are, independently, 0, 1, or 2.

In certain embodiments, the present invention provides a polypeptide ofthe formulae:

wherein K, M, L₁, L₂, R^(a), R^(b), R^(c), R^(e), R^(f), X_(AA), R^(KL),R^(LL), R^(LM), s, t, j, p, y, and z are as defined and described aboveand herein.

In certain embodiments, the present invention provides a polypeptide ofthe formulae:

wherein K, M, L₁, L₂, R^(a), R^(b), R^(c), R^(e), R^(f), X_(AA), R^(KL),R^(LL), R^(LM), s, t, j, p, y, and z are as defined and described aboveand herein.

In certain embodiments, the present invention provides a polypeptide ofthe formulae:

wherein K, M, L₁, L₂, R^(a), R^(b), R^(c), R^(e), R^(f), X_(AA), R^(KL),R^(LL), R^(LM), s, t, j, p, y, and z are as defined and described aboveand herein.

In certain embodiments, each instance of K, L₁, L₂, and M,independently, corresponds to a bond, cyclic or acyclic, branched orunbranched, substituted or unsubstituted C₁₋₂₀ alkylene; cyclic oracyclic, branched or unbranched, substituted or unsubstituted C₁₋₂₀alkenylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted C₁₋₂₀ alkynylene; cyclic or acyclic, branched orunbranched, substituted or unsubstituted C₁₋₂₀ heteroalkylene; cyclic oracyclic, branched or unbranched, substituted or unsubstituted C₁₋₂₀heteroalkenylene; cyclic or acyclic, branched or unbranched, substitutedor unsubstituted C₁₋₂₀ heteroalkynylene; substituted or unsubstitutedC₁₋₂₀ arylene; substituted or unsubstituted C₁₋₂₀ heteroarylene; orsubstituted or unsubstituted C₁₋₂₀ acylene; cyclic or acyclic, branchedor unbranched, substituted or unsubstituted C₁₋₁₅ alkylene; cyclic oracyclic, branched or unbranched, substituted or unsubstituted C₁₋₅alkenylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted C₁₋₅ alkynylene; cyclic or acyclic, branched orunbranched, substituted or unsubstituted C₁₋₁₅ heteroalkylene; cyclic oracyclic, branched or unbranched, substituted or unsubstituted C₁₋₁₅heteroalkenylene; cyclic or acyclic, branched or unbranched, substitutedor unsubstituted C₁₋₁₅ heteroalkynylene; substituted or unsubstitutedC₁₋₁₅ arylene; substituted or unsubstituted C₁₋₁₅ heteroarylene; orsubstituted or unsubstituted C₁₋₁₅ acylene; cyclic or acyclic, branchedor unbranched, substituted or unsubstituted C₁₋₁₀ alkylene; cyclic oracyclic, branched or unbranched, substituted or unsubstituted C₁₋₁₀alkenylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted C₁₋₁₀ alkynylene; cyclic or acyclic, branched orunbranched, substituted or unsubstituted C₁₋₁₀ heteroalkylene; cyclic oracyclic, branched or unbranched, substituted or unsubstituted C₁₋₁₀heteroalkenylene; cyclic or acyclic, branched or unbranched, substitutedor unsubstituted C₁₋₁₀ heteroalkynylene; substituted or unsubstitutedC₁₋₁₀ arylene; substituted or unsubstituted C₁₋₁₀ heteroarylene; orsubstituted or unsubstituted C₁₋₁₀ acylene; cyclic or acyclic, branchedor unbranched, substituted or unsubstituted C₁₋₈ alkylene; cyclic oracyclic, branched or unbranched, substituted or unsubstituted C₁₋₈alkenylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted C₁₋₈ alkynylene; cyclic or acyclic, branched orunbranched, substituted or unsubstituted C₁₋₈ heteroalkylene; cyclic oracyclic, branched or unbranched, substituted or unsubstituted C₁₋₈heteroalkenylene; cyclic or acyclic, branched or unbranched, substitutedor unsubstituted C₁₋₈ heteroalkynylene; substituted or unsubstitutedC₁₋₈ arylene; substituted or unsubstituted C₁₋₈ heteroarylene; orsubstituted or unsubstituted C₁₋₈ acylene; cyclic or acyclic, branchedor unbranched, substituted or unsubstituted C₁₋₅ alkylene; cyclic oracyclic, branched or unbranched, substituted or unsubstituted C₁₋₅alkenylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted C₁₋₅ alkynylene; cyclic or acyclic, branched orunbranched, substituted or unsubstituted C₁₋₅ heteroalkylene; cyclic oracyclic, branched or unbranched, substituted or unsubstituted C₁₋₅heteroalkenylene; cyclic or acyclic, branched or unbranched, substitutedor unsubstituted C₁₋₅ heteroalkynylene; substituted or unsubstitutedC₁₋₅ arylene; substituted or unsubstituted C₁₋₅ heteroarylene; orsubstituted or unsubstituted C₁₋₅ acylene.

In certain embodiments, K is acyclic. In certain embodiments, K isunbranched. In certain embodiments, K is unsubstituted. In certainembodiments, K is a bond. In certain embodiments, K is not a bond.

In certain embodiments, M is acyclic. In certain embodiments, M isunbranched. In certain embodiments, M is unsubstituted. In certainembodiments, M is a bond. In certain embodiments, M is not a bond.

In certain embodiments, L₁ is acyclic. In certain embodiments, L₁ isunbranched. In certain embodiments, L₁ is unsubstituted. In certainembodiments, L₁ is a bond. In certain embodiments, L₁ is not a bond.

In certain embodiments, L₂ is acyclic. In certain embodiments, L₂ isunbranched. In certain embodiments, L₂ is unsubstituted. In certainembodiments, L₂ is a bond. In certain embodiments, L₂ is not a bond.

In certain embodiments, L₁ and L₂ are the same. In certain embodiments,L₁ and L₂ are different. In certain embodiments, when L₁ is a bond, L₂is not a bond, or when L₂ is a bond, L₁ is not a bond. In certainembodiments, a polypeptide of any of the above formulae wherein L₁ andL₂ are both bonds is specifically excluded.

In certain embodiments, K and M are the same. In certain embodiments, Kand M are different.

In certain embodiments, K and L₁ are the same. In certain embodiments, Kand L₁ are different. In certain embodiments, K and L₂ are the same. Incertain embodiments, K and L₂ are different.

In certain embodiments, M and L₁ are the same. In certain embodiments, Mand L₁ are different. In certain embodiments, M and L₂ are the same. Incertain embodiments, M and L₂ are different.

In certain embodiments, all of K, L₁, L₂, and M are the same. In certainembodiments, all of K, L₁, L₂, and M are different.

In certain embodiments, each instance of K, L₁, L₂, and M,independently, corresponds to the formulae: —(CH₂)_(g+1)—;—(CH₂)_(g)—S—(CH₂)_(g)—; —(CH₂)_(g)(C═O)—S—(CH₂)_(g)—;—(CH₂)_(g)O—(CH₂)_(g)—; —(CH₂)_(g)—(C═O)—O—(CH₂)_(g)—;—(CH₂)_(g)—NH—(CH₂)_(g)—; —(CH₂)_(g)—(C═O)—NH—(CH₂)_(g)—;—(CH₂)_(g)CH(CH₃)—O—(CH₂)_(g)—;

wherein each instance of g is, independently, 0 to 10, inclusive.

In certain embodiments, each instance of K, L₁, L₂, and M,independently, corresponds to the formulae —(CH₂)_(g+1)—, and g is 0, 1,2, 3, 4, 5, or 6.

In certain embodiments, —[X_(AA)]— corresponds to the formulae:

wherein:each instance of R and R′ are, independently, hydrogen, or a suitableamino acid side chain as defined herein, and R^(a) is as previouslydefined above and herein.

Suitable amino acid side chains include, but are not limited to, bothnatural and unnatural amino acid side chains as provided in Tables 1 to3, and as described herein. In certain embodiments, each instance ofX_(AA) is an alpha amino acid, corresponding to the formula (a). Incertain embodiments, each instance of X_(AA) is a natural L-amino acid,as provided in Table 1. In certain embodiments, each instance of X_(AA)is, independently, a natural L-amino acid as provided in Table 1, or anunnatural D-amino acid as provided in Table 2.

The group R^(e) corresponds to the C-terminus of the peptide chain, andcorresponds to the variables —R^(E), —OR^(E), —N(R^(E))₂, or —SR^(E),wherein R^(E) is as defined above and herein. For example, if —[X_(AA)]—corresponds to an alpha amino acid of formula:

it follows that, in certain embodiments, —[X_(AA)]_(t)—R^(e) correspondsto the formulae:

wherein each instance of R^(E) is, independently, hydrogen; cyclic oracyclic, branched or unbranched, substituted or unsubstituted aliphatic;cyclic or acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; substituted or unsubstituted acyl; a resin; ora suitable hydroxyl, amino, or thiol protecting group; and two R^(E)groups taken together may optionally form a substituted or unsubstituted5- to 6-membered heterocyclic or heteroaromatic ring.

In certain embodiments, R^(e) is —OR^(E), and R^(E) is hydrogen, cyclicor acyclic, branched or unbranched, substituted or unsubstitutedaliphatic; cyclic or acyclic, branched or unbranched, substituted orunsubstituted heteroaliphatic; substituted or unsubstituted aryl;substituted or unsubstituted heteroaryl; substituted or unsubstitutedacyl; a resin; or a suitable hydroxyl protecting group.

In certain embodiments, R^(e) is —SR^(E), and R^(E) is hydrogen, cyclicor acyclic, branched or unbranched, substituted or unsubstitutedaliphatic; cyclic or acyclic, branched or unbranched, substituted orunsubstituted heteroaliphatic; substituted or unsubstituted aryl;substituted or unsubstituted heteroaryl; substituted or unsubstitutedacyl; a resin; or a suitable thiol protecting group.

In certain embodiments, R^(e) is —N(R^(E))₂, and each instance of R^(E)is, independently, hydrogen, cyclic or acyclic, branched or unbranched,substituted or unsubstituted aliphatic; cyclic or acyclic, branched orunbranched, substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; substitutedor unsubstituted acyl; a resin; a suitable amino protecting group; ortwo R^(E) groups together form a substituted or unsubstituted 5- to6-membered heterocyclic or heteroaromatic ring.

The group R^(f) corresponds to the N-terminus of the peptide chain. Forexample, if —[X_(AA)]— corresponds to an alpha amino acid of formula:

it follows that, in certain embodiments, R^(f)—[X_(AA)]^(s)— correspondsto the formulae:

wherein R and R′ are defined as above and herein; andwherein R^(f) is hydrogen; cyclic or acyclic, branched or unbranched,substituted or unsubstituted aliphatic; cyclic or acyclic, branched orunbranched, substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; substitutedor unsubstituted acyl; a resin; a suitable amino protecting group; alabel optionally joined by a linker, wherein the linker is selected fromcyclic or acyclic, branched or unbranched, substituted or unsubstitutedalkylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted alkenylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted alkynylene; cyclic or acyclic, branched orunbranched, substituted or unsubstituted heteroalkylene; cyclic oracyclic, branched or unbranched, substituted or unsubstitutedheteroalkenylene; cyclic or acyclic, branched or unbranched, substitutedor unsubstituted heteroalkynylene; substituted or unsubstituted arylene;substituted or unsubstituted heteroarylene; or substituted orunsubstituted acylene; or R^(f) and R^(a) together form a substituted orunsubstituted 5- to 6-membered heterocyclic or heteroaromatic ring.

In certain embodiments, R^(f) is hydrogen. In certain embodiments, R^(f)is C₁₋₆ alkyl. In certain embodiments, R^(f) is —CH₃. In certainembodiments, R^(f) is a suitable amino protecting group. In certainembodiments, R^(f) is -Boc. In certain embodiments, R^(f) is -Fmoc. Incertain embodiments, R^(f) is acyl. In certain embodiments, R^(f) is—(C═O)CH₃.

In certain embodiments, R^(f) is a label optionally joined by a linker,wherein the linker is selected from cyclic or acyclic, branched orunbranched, substituted or unsubstituted alkylene; cyclic or acyclic,branched or unbranched, substituted or unsubstituted alkenylene; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedalkynylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted heteroalkylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroalkenylene; cyclic or acyclic,branched or unbranched, substituted or unsubstituted heteroalkynylene;substituted or unsubstituted arylene; substituted or unsubstitutedheteroarylene; or substituted or unsubstituted acylene.

Exemplary labels include, but are not limited to FITC and biotin:

In certain embodiments, the label is directly joined to the inventivepolypeptide (i.e., through a bond).

In certain embodiments, the label is indirectly joined to the inventivepolypeptide (i.e., through a linker).

In certain embodiments, the linker is a cyclic or acyclic, branched orunbranched, substituted or unsubstituted alkylene. In certainembodiments, the linker is a cyclic or acyclic, branched or unbranched,substituted or unsubstituted alkenylene. In certain embodiments, thelinker is a cyclic or acyclic, branched or unbranched, substituted orunsubstituted alkynylene. In certain embodiments, the linker is a cyclicor acyclic, branched or unbranched, substituted or unsubstitutedheteroalkylene. In certain embodiments, the linker is a cyclic oracyclic, branched or unbranched, substituted or unsubstitutedheteroalkenylene. In certain embodiments, the linker is a cyclic oracyclic, branched or unbranched, substituted or unsubstitutedheteroalkynylene. In certain embodiments, the linker is a substituted orunsubstituted arylene. In certain embodiments, the linker is asubstituted or unsubstituted heteroarylene. In certain embodiments, thelinker is a substituted or unsubstituted acylene.

For example, in certain embodiments, the linker is cyclic or acyclic,branched or unbranched, substituted or unsubstituted heteroalkyleneselected from:

In certain embodiments, R^(a) is hydrogen. In certain embodiments, R^(a)is C₁₋₆ alkyl. In certain embodiments, R^(a) is —CH₃. In certainembodiments, R^(a) is acyl. In certain embodiments, R^(a) is —(C═O)CH₃.

In certain embodiments, each instance of R^(b) is, independently,hydrogen or cyclic or acyclic, branched or unbranched, substituted orunsubstituted aliphatic. In certain embodiments, R^(b) is hydrogen or—CH₃. In certain embodiments, R^(b) is —CH₃.

In certain embodiments, each instance of R^(c), is, independently,hydrogen; cyclic or acyclic, branched or unbranched, substituted orunsubstituted aliphatic; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl. In certainembodiments, each instance of R^(e), is, independently, hydrogen; orcyclic or acyclic, branched or unbranched, substituted or unsubstitutedaliphatic. In certain embodiments, each instance of R^(e) is,independently, hydrogen or cyclic or acyclic, branched or unbranched,substituted or unsubstituted alkyl. In certain embodiments, R^(b) ishydrogen or —CH₃. In certain embodiments, each instance of R^(c) ishydrogen.

In certain embodiments, each instance of R^(KL), R^(LL), and R^(LM), is,independently, hydrogen; cyclic or acyclic, branched or unbranched,substituted or unsubstituted aliphatic; cyclic or acyclic, branched orunbranched, substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; substitutedor unsubstituted acyl; substituted or unsubstituted hydroxyl;substituted or unsubstituted thiol; substituted or unsubstituted amino;azido; cyano; isocyano; halo; or nitro.

In certain embodiments, each instance of R^(KL), R^(LL), and R^(LM), is,independently, hydrogen; cyclic or acyclic, branched or unbranched,substituted or unsubstituted aliphatic; cyclic or acyclic, branched orunbranched, substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; substitutedor unsubstituted acyl; substituted or unsubstituted hydroxyl;substituted or unsubstituted thiol; substituted or unsubstituted amino;cyano; isocyano; halo; or nitro.

In certain embodiments, p is 0. In certain embodiments, p is 1. Incertain embodiments, p is 2. In certain embodiments, p is 3. In certainembodiments, p is 4. In certain embodiments, p is 5. In certainembodiments, p is 6. In certain embodiments, p is 7. In certainembodiments, p is 8. In certain embodiments, p is 9. In certainembodiments, p is 10.

The variables y and z indicate how many amino acids, defined by thevariable [X_(AA)], there are between amino acids containing terminallyunsaturated amino acid side chain(s), as provided in polypeptides offormulae (I) to (VII). For example, as depicted below for a polypeptideof formula (I), wherein p is 0 (hereafter designated as formula (I-c)),wherein the variables K, M, L₁, L₂, R^(a), R^(b), R^(c), R^(e), R^(f),X_(AA), s, t, j, y, and z are as defined and described above and herein,and wherein i represents one site of an alpha,alpha-disubstituted(terminally unsaturated amino acid side chain) amino acid, variable yprovides information as to the position of the amino acid containing aterminally unsaturated side chain on the N-terminal side of i, such asthe positions i−3, i−4, i−6, and i−7, and z provides information as tothe position of the amino acid containing a terminally unsaturated sidechain on the C-terminal side of i, such as the positions i+3, i+4, i+6,and i+7. Table 3 correlates these specific locations of i relative tothe variables y and z for formula (I-c).

TABLE 3 i − 7 i − 6 i − 4 i − 3 i i + 3 i + 4 i + 6 i + 7 y 6 5 3 2 z 23 5 6

In certain embodiments, each instance of y and z are, independently, 2,3, 5, or 6.

In certain embodiments, both y and z are 2. In certain embodiments, bothy and z are 3. In certain embodiments, both y and z are 5. In certainembodiments, both y and z are 6.

In certain embodiments, y is 2 and z is 3. In certain embodiments, y is2 and z is 5. In certain embodiments, y is 2 and z is 6.

In certain embodiments, y is 3 and z is 2. In certain embodiments, y is3 and z is 5. In certain embodiments, y is 3 and z is 6.

In certain embodiments, y is 5 and z is 2. In certain embodiments, y is5 and z is 3. In certain embodiments, y is 5 and z is 6.

In certain embodiments, y is 6 and z is 2. In certain embodiments, y is6 and z is 3. In certain embodiments, y is 6 and z is 5.

In certain embodiments, the present invention also providesintermediates used in the synthesis of inventive polypeptides. Forexample, the present invention provides bis-amino acids of formula:

wherein L₁, L₂, R^(a), R^(c), R^(e), R^(f), x, and

are as defined and described above and herein.

In certain embodiments, a bis amino acid of formula (A) has the formula:

wherein L₁, L₂, R^(a), R^(c), R^(e), and R^(f) are as defined anddescribed above and herein.

In certain embodiments, a bis amino acid of formula (A) has the formula:

wherein L₁, L₂, R^(a), R^(e), and R^(f) are as defined and describedabove and herein.

Exemplary amino acids of formula (A) include, but are not limited to,those as depicted below, wherein R^(a), R^(f), and R^(e) are definedabove and herein. In certain embodiments, R^(a) is hydrogen, and R^(f)is a suitable amino protecting group. In certain embodiments, R^(a) ishydrogen, and R^(f) is -Boc or -Fmoc. In certain embodiments, both R^(a)and R^(f) are suitable amino protecting groups. In certain embodiments,both R^(a) and R^(f) are hydrogen. In certain embodiments, R^(e) ishydrogen.

Exemplary Amino Acids of Formula (A).

Methods of Synthesis

The present invention is also directed to methods of synthesizingstitched and unstitched inventive polypeptides.

The synthesis of an inventive polypeptide first involves the selectionof a desired sequence and number of amino acids and amino acidanalogues. As one of ordinary skill in the art will realize, the number,stereochemistry, and type of amino acid structures (natural ornon-natural) selected will depend upon the size of the polypeptide to beprepared, the ability of the particular amino acids to generate adesired structural motif (e.g., an alpha-helix), and any particularmotifs that are desirable to mimic (for example, a p53 donor helicalpeptide).

Once the amino acids are selected, synthesis of the inventivepolypeptide can be achieved using standard deprotection and couplingreactions. Formation of peptide bonds and polypeptide synthesis aretechniques well-known to one skilled in the art, and encompass bothsolid phase and solution phase methods; see generally, Bodanszky andBodanszky, The Practice of Peptide Synthesis, Springer-Verlag, Berlin,1984; Atherton and Sheppard, Solid Phase Peptide Synthesis: A PracticalApproach, IRL Press at Oxford University Press Oxford, England, 1989,and Stewart and Young, Solid phase Peptide Synthesis, 2nd edition,Pierce Chemical Company, Rockford, 1984, the entire contents of each ofwhich are incorporated herein by reference. In both solution phase andsolid phase techniques, the choice of the protecting groups must beconsidered, as well as the specific coupling techniques to be utilized.For a detailed discussion of peptide synthesis techniques for solutionphase and solid phase reactions, see, Bioorganic chemistry: Peptides andProteins, Hecht, Oxford University Press, New York: 1998, the entirecontents of which are incorporated herein by reference.

In certain embodiments, the method comprises a solution phase synthesisof an inventive polypeptide. Solution phase synthesis, as mentionedabove, is a well-known technique for the construction of polypeptides.An exemplary solution phase synthesis comprises the steps of: (1)providing an amino acid protected at the N-terminus with a suitableamino protecting group; (2) providing an amino acid protected at theC-terminus with a suitable carboxylic acid protecting group; (3)coupling the N-protected amino acid to the C-protected amino acid; (4)deprotecting the product of the coupling reaction; and (5) repeatingsteps (3) to (4) until a desired polypeptide is obtained, wherein atleast two of the amino acids coupled at any of the above steps eachcomprise at least one terminally unsaturated amino acid sidechain, andat least one α,α-disubstituted amino acid comprises two terminallyunsaturated amino acid side chains. During the course of the abovesynthesis, various parameters can be varied, including, but not limitedto placement of amino acids with terminally unsaturated side chains,stereochemistry of amino acids, terminally unsaturated side chain lengthand functionality, and amino acid residues utilized.

In certain embodiments, the method comprises a solid phase synthesis ofan inventive polypeptide. Solid phase synthesis, as mentioned above, isa well-known technique for the construction of polypeptides. Anexemplary solid phase synthesis comprises the steps of: (1) providing aresin-bound amino acid; (2) deprotecting the resin bound amino acid; (3)coupling an amino acid to the deprotected resin-bound amino acid; (4)repeating steps (3) until a desired peptide is obtained, wherein atleast two of the amino acids coupled at any of the above steps eachcomprise at least one terminally unsaturated amino acid sidechain, andat least one α,α-disubstituted amino acid comprises two terminallyunsaturated amino acid side chains. During the course of the abovesynthesis, various parameters can be varied, including, but not limitedto placement of amino acids with terminally unsaturated side chains,stereochemistry of amino acids, terminally unsaturated side chain lengthand functionality, and amino acid residues utilized.

After a desired polypeptide is synthesized using an appropriatetechnique, the polypeptide is contacted with a specific catalyst topromote “stitching” of the polypeptide. For example, the resin-boundpolypeptide may be contacted with a catalyst to promote “stitching,” ormay first be cleaved from the resin, and then contacted with a catalystto promote “stitching.”

Thus, in one aspect, the present invention is directed to a method ofmaking a polypeptide of formulae (I), (I-a), (I-b), or (I-c) comprisingthe steps of:

-   -   (i) providing a bis-amino acid of the formula:

-   -   (ii) providing an amino acid of the formula:

-   -   (iii) providing an amino acid of the formula:

wherein the variables K, L₁, L₂, M, R^(a), R^(b), R^(c), R^(e), R^(f),x, and

are defined herein;

-   -   (iv) providing at least one additional amino acid; and    -   (v) coupling said amino acids of formulae (A), (B), and (C) with        at least one amino acid of step (iv) under suitable conditions        to provide a polypeptide of formulae (I), (I-a), (I-b), or        (I-c).

As used herein, the phrase “providing at least one additional aminoacid” refers to providing at least one natural or unnatural amino acidstructurally different than a compound of formulae (A), (B), or (C). Theabove synthetic method may employ any and all known amino acids in orderto generate a polypeptide of any one of formulae (I) to (VII), andsubsets thereof. In certain embodiments, the amino acids employable bythe above synthetic method are defined and described herein.

In certain embodiments, step (iv) provides at least two additional(i.e., structurally different) amino acids. In certain embodiments, step(iv) provides at least three additional amino acids. In certainembodiments, step (iv) provides at least four additional amino acids. Incertain embodiments, step (iv) provides at least five additional aminoacids.

In certain embodiments, step (iv) further includes providing a peptidewhich will be incorporated into the inventive polypeptide. In certainembodiments, step (iv) further includes providing a peptide comprisingat least 2 amino acids. In certain embodiments, step (iv) furtherincludes providing a peptide comprising at least 3 amino acids. Incertain embodiments, step (iv) further includes providing a peptidecomprising at least 4 amino acids. In certain embodiments, step (iv)further includes providing a peptide comprising at least 5 amino acids.

In certain embodiments, the at least one type of additional amino acidof step (iv) corresponds to the formulae:

wherein R′, R, R^(a), R^(e), and R^(f) are defined above and herein.

Different amino acids have different propensities for forming differentsecondary structures. For example, methionine (M), alanine (A), leucine(L), glutamate (E), and lysine (K) all have especially high alpha-helixforming propensities. In contrast, proline (P) and glycine (G) arealpha-helix disruptors. Thus, in certain embodiments, the at least oneamino acid of step (iv) refers to a group selected from alanine,arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,serine, threonine, tryptophan, tyrosine, and valine.

In certain embodiments, the above reaction of step (iv) furthercomprises the use of a coupling reagent. Exemplary coupling reagentsinclude, but are not limited to,benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate(BOP), benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyBOP), bromo-tris-pyrrolidino phosphoniumhexafluorophosphate (PyBroP), 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide (EDC), N,N′-carbonyldiimidazole (CDI),3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT),1-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxy-7-benzotriazole (HOBt),2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU),2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU),2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TATU),2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU),N,N,N′,N′-tetramethyl-O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)uraniumtetrafluoroborate (TDBTU), and O—(N-succinimidyl)-1,1,3,3-tetramethyluranium tetrafluoroborate (TSTU)).

In certain embodiments, the above reaction of step (iv) furthercomprises a suitable base. Suitable bases include, but are not limitedto, potassium carbonate, potassium hydroxide, sodium hydroxide,tetrabutylammonium hydroxide, benzyltrimethylammonium hydroxide,triethylbenzylammonium hydroxide, 1,1,3,3-tetramethylguanidine,1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), N-methylmorpholine,diisopropylethylamine (DIPEA), tetramethylethylenediamine (TMEDA),pyridine (Py), 1,4-diazabicyclo[2.2.2] octane (DABCO), N,N-dimethylaminopyridine (DMAP), or triethylamine (NEt₃).

In certain embodiments, the reaction of step (iv) is carried out in asuitable medium. A suitable medium is a solvent or a solvent mixturethat, in combination with the combined reacting partners and reagents,facilitates the progress of the reaction therebetween. A suitablesolvent may solubilize one or more of the reaction components, or,alternatively, the suitable solvent may facilitate the suspension of oneor more of the reaction components; see generally, March's AdvancedOrganic Chemistry: Reactions, Mechanisms, and Structure, M. B. Smith andJ. March, 5^(t) Edition, John Wiley & Sons, 2001, and ComprehensiveOrganic Transformations, R. C. Larock, 2^(nd) Edition, John Wiley &Sons, 1999, the entire contents of each of which are incorporated hereinby reference. Suitable solvents for include ethers, halogenatedhydrocarbons, aromatic solvents, polar aprotic solvents, or mixturesthereof. In other embodiments, the solvent is diethyl ether, dioxane,tetrahydrofuran (THF), dichloromethane (DCM), dichloroethane (DCE),acetonitrile (ACN), chloroform, toluene, benzene, dimethylformamide(DMF), dimethylacetamide (DMA), dimethylsulfoxide (DMSO), N-methylpyrrolidinone (NMP), or mixtures thereof.

In other embodiments, the reaction of step (iv) is conducted at suitabletemperature, such as between about 0° C. and about 100° C.

The present invention is also directed to a method of making apolypeptide of formulae (II), (III), (IV), (V), (VI), or (VII), or anysubsets thereof, comprising the steps of:

-   -   (i) providing a bis-amino acid of the formula:

-   -   (ii) providing an amino acid of the formula:

-   -   (iii) providing an amino acid of the formula:

wherein K, L₁, L₂, M, R^(a), R^(b), R^(c), R^(e), R^(f), x, and

are defined above and herein;

-   -   (iv) providing at least one additional amino acid;    -   (v) coupling said amino acids of formulae (A), (B), and (C) with        at least one additional amino acid of step (iv) to provide a        polypeptide of formulae (I), (I-a), or (I-b); and    -   (vi) treating the polypeptide of step (v) with a catalyst.

In certain embodiments, the reaction of step (iv) comprises a suitablecoupling reagent, a suitable base, a suitable medium, and/or isconducted at a suitable temperature.

One of ordinary skill in the art will realize that a variety ofcatalysts can be utilized in step (vi) of the above method. Selection ofa particular catalyst will vary with the reaction conditions utilizedand the functional groups present in the particular peptide. In certainembodiments, the catalyst of step (vi) is a ring closing metathesis(RCM) catalyst. In certain embodiments, the RCM catalyst is a tungsten(W), molybdenum (Mo), or ruthenium (Ru) catalyst. In certainembodiments, the RCM catalyst is a ruthenium catalyst. Suitable RCMcatalysts employable by the above synthetic method include catalysts areas depicted below, and as described in see Grubbs et al., Acc. Chem.Res. 1995, 28, 446-452; U.S. Pat. No. 5,811,515; Schrock et al.,Organometallics (1982) 1 1645; Gallivan et al., Tetrahedron Letters(2005) 46:2577-2580; Furstner et al., J. Am. Chem. Soc. (1999) 121:9453;and Chem. Eur. J. (2001) 7:5299; the entire contents of each of whichare incorporated herein by reference.

In certain embodiments, the RCM catalyst is a Schrock catalyst. Incertain embodiments, the Schrock catalyst is selected from any of thefollowing:

In certain embodiments, the RCM catalyst is a Grubbs catalyst. Incertain embodiments, the Grubbs catalyst is selected from any of thefollowing:

X═Cl; Br; I

Cy=cyclohexyl

Benzylidenebis-(tricyclohexylphosphine)-dichlororuthenium (X═Cl)

Benzylidenebis-(tricyclohexylphosphine)-dibromoruthenium (X═Br)

Benzylidenebis-(tricyclohexylphosphine)-diiodoruthenium (X═I);

X═Cl; Br; I

R=cyclohexyl (Cy); phenyl (Ph); benzyl (Bn)

1,3-(Bis(mesityl)-2-imidazolidinylidene)dichloro-(phenylmethylene)(tricyclohexyl-phosphine)ruthenium (X═Cl; R=cyclohexyl)

1,3-(Bis(mesityl)-2-imidazolidinylidene)dibromo-(phenylmethylene)(tricyclohexyl-phosphine)ruthenium (X═Br; R=cyclohexyl)

1,3-(Bis(mesityl)-2-imidazolidinylidene)diiodo-(phenylmethylene)(tricyclohexyl-phosphine)ruthenium (X═I; R=cyclohexyl)

1,3-(Bis(mesityl)-2-imidazolidinylidene)dichloro-(phenylmethylene)(triphenylphosphine)ruthenium (X═Cl; R=phenyl)

1,3-(Bis(mesityl)-2-imidazolidinylidene)dichloro-(phenylmethylene)(tribenzylphosphine)ruthenium (X═Cl; R=benzyl);

In certain embodiments, the RCM catalyst is a Grubbs-Hoveyda catalyst.In certain embodiments, the Grubbs-Hoveyda catalyst is selected from anyof the following:

In certain embodiments, the RCM catalyst is selected from any of thefollowing:

It will also be appreciated, that in addition to RCM catalysts, otherreagents capable of promoting carbon-carbon bond formation can also beutilized. For example, other reactions that can be utilized, include,but are not limited to palladium coupling reactions, transition metalcatalyzed cross coupling reactions, pinacol couplings (terminalaldehydes), hydrozirconation (terminal alkynes), nucleophilic additionreactions, and NHK (Nozaki-Hiyama-Kishi (Furstner et al., J. Am. Chem.Soc. 1996, 118, 12349)) coupling reactions. Thus, the appropriatereactive moieties are first incorporated into desired amino acids orunnatural amino acids, and then the peptide is subjected to reactionconditions to effect “stitching” and subsequent stabilization of adesired secondary structure.

In certain embodiments, a compound of formula (B) has the formula:

wherein K, R^(a), R^(c), R^(e), and R^(f) are defined above and herein.

In certain embodiments, a compound of formula (B) has the formula:

wherein K, R^(a), R^(c), R^(e), and R^(f) are defined above and herein.

In certain embodiments, a compound of formula (C) has the formula:

wherein M, R^(a), R^(c), R^(e), and R^(f) are defined above and herein.

In certain embodiments, a compound of formula (C) has the formula:

wherein M, R^(a), R^(c), R^(e), and R^(f) are defined above and herein.

Exemplary amino acids of formulae (B) and (C) (corresponding to aminoacids with one terminally unsaturated side chain) include, but are notlimited to, those as depicted below, wherein R^(a), R^(f), and R^(e) aredefined above and herein. In certain embodiments, R^(a) is hydrogen, andR^(f) is -Boc or -Fmoc. In certain embodiments, both R^(a) and R^(f) arehydrogen. In certain embodiments, R^(e) is hydrogen.

In certain embodiments, an amino acid of formula (B) is anR-configurated amino acids. In certain embodiments, an R-configuratedamino acid of formula (B) is a D-amino acid. In certain embodiments, anamino acid of formula (B) is an S-configurated amino acids. In certainembodiments, an S-configurated amino acid of formula (B) is an L-aminoacid. In certain embodiments, an amino acid of formula (B) is racemic.In certain embodiments, amino acids of formula (B) are a mixture of D-and L-amino acids.

In certain embodiments, an amino acid of formula (C) is anR-configurated amino acid. In certain embodiments, an R-configuratedamino acid of formula (C) is a D-amino acid. In certain embodiments, anamino acid of formula (C) is an S-configurated amino acid. In certainembodiments, an S-configurated amino acid of formula (C) is an L-aminoacid. In certain embodiments, an amino acid of formula (C) is racemic.In certain embodiments, amino acids of formula (C) are a mixture of D-and L-amino acids.

Exemplary Amino Acids of Formulae (B) and (C)

In another aspect, the present invention provides a method ofsynthesizing an inventive polypeptide comprising the steps of:

(1) providing a selected number of amino acids comprising (i) at leasttwo amino acids, each comprising at least one terminally unsaturatedamino acid sidechain, and (ii) at least one α,α-disubstituted amino acidcomprising two terminally unsaturated amino acid side chains;

(2) coupling the selected number of amino acids together to generate afirst peptide; and

(3) treating the first peptide with a suitable catalyst to provide astitched peptide.

In certain embodiments, divinyl amino acid as “an α,α-disubstitutedamino acid comprising two terminally unsaturated amino acid side chains”is specifically excluded.

In certain embodiments, each terminally unsaturated amino acid sidechainis reactive toward ring closing metathesis. In certain embodiments, thesuitable catalyst is a ring metathesis catalyst. In certain embodiments,the ring closing metathesis catalyst may generate at least twocross-linked rings by the above method. Depending upon the nature of theselected amino acids and their specific location in the peptide chain,stitched peptides of the present invention may comprise at least 2, 3,4, 5, 6, or 7, cross-links, and may comprise one or moreconstitutional/structural isomers (i.e., compounds with the samemolecular weight but having different connectivity). For example, asdepicted in the following Scheme, in certain embodiments, tandem“stitching” of a polypeptide of formula (I-c), as described above andherein, provides three possible stitched products designated herein as(II-d), (VIII), and (IX), wherein K, M, L₁, L₂, R^(a), R^(b), R^(c),R^(e), R^(f), X_(AA), R^(KL), R^(LL), R^(LM), s, t, j, p, y, z, u, q,and v, are a as defined herein.

In certain embodiments, the above synthetic method generates onestitched product as a preferred product. As used herein a “preferredproduct” refers to one constitutional isomer present as the majorconstituent in a mixture of isomers. In certain embodiments, a“preferred product” refers to one constitutional isomer present as acomponent in at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, or 99%, of an isomeric mixture. In certain embodiments, thepreferred product corresponds to a compound of formula (II-d).

In certain embodiments, nested (e.g., formula (VIII)) or overlappling(e.g., formula (IX)) cross-linked products are minor products. Incertain embodiments, nested (e.g., formula (VIII)) or overlappling(e.g., formula (IX)) cross-linked products are not generated from thereaction.

Tandem “Stitching” of a Polypeptide of Formula (I-c)

The above synthetic method may be further modified to include at leastthree cross-linking staples by:

(1) providing a selected number of natural or unnatural amino acids,wherein said number comprises: (i) at least four amino acids, eachcomprising at least one terminally unsaturated amino acid sidechain, and(ii) at least one α,α-disubstituted amino acid comprising two terminallyunsaturated amino acid side chains;

(2) coupling the selected number of amino acids together to generate afirst peptide; and

(3) treating the first peptide with a suitable catalyst.

Additionally, the above synthetic method may be modified to include atleast three cross-linking staples by:

(1) providing a selected number of natural or unnatural amino acids,wherein said number comprises: (i) at least two amino acids, eachcomprising at least one terminally unsaturated amino acid sidechain, and(ii) at least two α,α-disubstituted amino acids, each comprising twoterminally unsaturated amino acid side chains;

(2) coupling the selected number of amino acids together to generate afirst peptide; and

(3) treating the first peptide with a suitable catalyst.

The above modifications to the synthetic method are provided as examplesonly, and are not intended to limit the scope or intent of the presentinvention. The present invention contemplates any and all types ofmodifications in order to provide at least 2, 3, 4, 5, 6, or 7,cross-linked staples into the above described polypeptides.

The above amino acids comprising one to two terminally unsaturated aminoacid sidechains are so incorporated into the polypeptide chain in orderto provide proximal terminally unsaturated sidechains. These proximalterminally unsaturated sidechains may be in the same plane as, or sameside of the polypeptide chain as, each other in any given conformationof the polypeptide. Upon treatment with a suitable catalyst, theseproximal side chains react with each other via “stapling” to provide astitched, conformationally stabilized, polypeptide. In certainembodiments, the proximal terminally unsaturated sidechains are arrangedsuch that the resulting “staple” does not interfere with thebiological/therapeutic activity of the stitched inventive polypeptide.

Additional Synthetic Modifications

After “stitching” of an inventive polypeptide, as described above, themethod may further comprise additional synthetic modification(s). Anychemical or biological modification may be made. In certain embodiments,such modifications include reduction, oxidation, and nucleophilic orelectrophilic additions to a functional group (e.g., a double bondprovided from a metathesis reaction) of the cross-link to provide asynthetically modified stitched polypeptide. Other modifications mayinclude conjugation of a stitched polypeptide, or a syntheticallymodified stitched polypeptide, with a biologically active agent, labelor diagnostic agent anywhere on the stitched polypeptide scaffold, e.g.,such as at the N-terminus of the stitched polypeptide, the C-terminus ofthe stitched polypeptide, on an amino acid side chain of the stitchedpolypeptide, or at one or more modified or unmodified stitched sites(i.e., to a staple). Such modification may be useful in delivery of thepeptide or biologically active agent to a cell, tissue, or organ. Suchmodifications may allow for targeting to a particular type of cell ortissue.

Thus, in certain embodiments, the above synthetic method furthercomprises:

(vii) treating the polypeptide of step (vi) with a suitably reactiveagent under suitable conditions to provide a synthetically modifiedstitched polypeptide.

One of ordinary skill in the art will appreciate that a wide variety ofreactions, conditions, and “suitably reactive agent(s)” may be employedto promote such a transformation, therefore, a wide variety ofreactions, conditions, and reactive agents are envisioned; seegenerally, March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, M. B. Smith and J. March, 5^(th) Edition, John Wiley &Sons, 2001; Advance Organic Chemistry, Part B: Reactions and Synthesis,Carey and Sundberg, 3^(rd) Edition, Plenum Press, New York, 1993; andComprehensive Organic Transformations, R. C. Larock, 2^(nd) Edition,John Wiley & Sons, 1999, the entirety of each of which is herebyincorporated herein by reference. Exemplary “suitably reactive agents”may be any agent reactive with a multiple bond (e.g., a double or triplebond). In certain embodiments, suitably reactive agents are able toreact with a double bond or triple bond, for example, via ahydrogenation, osmylation, hydroxylation (mono- or di-), amination,halogenation, cycloaddition (e.g., cyclopropanation, aziridination,epoxidation), oxy-mercuration, and/or a hydroboronation reaction, toprovide a functionalized single bond or double bond. As one of ordinaryskill in the art will clearly recognize, these above-describedtransformations will introduce functionalities compatible with theparticular stabilized structures and the desired biologicalinteractions; such functionalities include, but are not limited to,hydrogen, cyclic or acyclic, branched or unbranched, substituted orunsubstituted aliphatic; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; substitutedor unsubstituted acyl; substituted or unsubstituted hydroxyl;substituted or unsubstituted amino; substituted or unsubstituted thiol,halo; cyano; nitro; azido; imino; oxo; and thiooxo.

In another aspect, in certain embodiments, the above method furthercomprises

-   -   (vii) treating the polypeptide of step (vi) with a suitably        reactive agent to provide a synthetically modified stitched        polypeptide, and    -   (viii) treating the modified stitched polypeptide of step (vii)        with a biologically active agent to provide a modified stitched        polypeptide conjugated to a biologically-active agent.

Furthermore, in another aspect, in certain embodiments, the above methodcomprises:

-   -   (vii) treating a stitched peptide of step (vi) with a        biologically active agent to provide a stitched peptide        conjugated to a biologically-active agent.

In another aspect, in certain embodiments, the above method furthercomprises

-   -   (vii) treating the polypeptide of step (vi) with a suitable        reagent to provide a synthetically modified stitched        polypeptide, and    -   (viii) treating the modified stitched polypeptide of step (vii)        with a diagnostic agent to provide a modified stitched        polypeptide conjugated to a diagnostic agent.

Furthermore, in another aspect, in certain embodiments, the above methodcomprises:

-   -   (vii) treating a stitched peptide of step (vi) with a diagnostic        agent to provide a stitched peptide conjugated to a diagnostic        agent.

Conjugation of an agent (e.g., a label, a diagnostic agent, abiologically active agent) to the inventive polypeptide may be achievedin a variety of different ways. The agent may be covalently conjugated,directly or indirectly, to the polypeptide at the site of stapling, orto the N-terminus or the C-terminus of the polypeptide chain.Alternatively, the agent may be noncovalently conjugated, directly orindirectly, to the polypeptide at the site of stapling, or to theN-terminus or the C-terminus of the polypeptide chain. Indirect covalentconjugation is by means of one or more covalent bonds. Indirectnoncovalent conjugation is by means of one or more noncovalent bonds.Conjugation may also be via a combination of non-covalent and covalentforces/bonds. The agent may also be conjugated through a covalent ornon-covalent linking group.

Any suitable bond may be used in the conjugation of a biologicallyactive agent and/or diagnostic agent to the inventive polypeptidepresent invention. Such bonds include amide linkages, ester linkages,disulfide linkages, carbon-carbon bonds, carbamate, carbonate, urea,hydrazide, and the like. In some embodiments, the bond is cleavableunder physiological conditions (e.g., enzymatically cleavable, cleavablewith a high or low pH, with heat, light, ultrasound, x-ray, etc).However, in some embodiments, the bond is not cleavable.

Combinatorial Synthesis of Novel Stabilized Structures

It will also be appreciated by one of ordinary skill in the art that thesynthetic method as described above can also be applied to combinatorialsynthesis of inventive polypeptides. Although combinatorial synthesistechniques can be applied in solution, it is more typical thatcombinatorial techniques are performed on the solid phase usingsplit-and-pool techniques. During the course of the combinatorialsynthesis, various parameters can be varied, including, but not limitedto placement of amino acids with terminally unsaturated side chains,stereochemistry of amino acids, terminally unsaturated side chain lengthand functionality, and amino acid residues utilized.

The present invention, in one aspect, provides methods for the synthesisof libraries of novel inventive polypeptides, as described above,comprising (1) providing a collection of resin-bound amino acids; (2)deprotecting each of said resin bound amino acids; (3) separating saidcollection of deprotected resin bound amino acids into n equal portions,wherein n represents the number of different types of amino acids to becoupled; (4) coupling of each of n types of amino acids to thedeprotected amino acid; (5) combining each of the n portions together;and (6) repeating steps (2)-(5) until a desired polypeptide is obtained,wherein at least two of the amino acids coupled at any of the abovesteps each comprise at least one terminally unsaturated amino acidsidechain, and at least one α,α-disubstituted amino acid comprises twoterminally unsaturated amino acid side chains. After a desiredpolypeptide is synthesized, the resin-bound polypeptide may be contactedwith a catalyst to promote “stitching,” or may first be cleaved from theresin, and then contacted with a catalyst to promote “stitching.”

It will be appreciated by one of ordinary skill in the art that thelibraries of compounds having stabilized secondary structures can befurther diversified at specific functional moieties after the desiredstabilized structures are formed. For example, free or latent amino acidfunctionalities may be diversified, or alternatively or additionally,free or latent functionality present on the cross-linkers may bediversified. In particularly preferred embodiments, in but one example,the hydrophilicity of stabilized structures may be increased by theintroduction of hydroxyl moieties. As one of ordinary skill in the artwill realize, the diversification reactions will be selected tointroduce functionalities compatible with the particular stabilizedstructures and the desired biological interactions, and thesefunctionalities include, but are not limited to hydrogen, cyclic oracyclic, branched or unbranched, substituted or unsubstituted aliphatic;cyclic or acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; substituted or unsubstituted acyl; substitutedor unsubstituted hydroxyl; substituted or unsubstituted amino;substituted or unsubstituted thiol, halo; cyano; nitro; azido; imino;oxo; and thiooxo.

Methods of Use

The present invention provides a method of treating a disease, disorder,or condition comprising administering to a subject diagnosed with orhaving susceptibility to the disease, disorder, or condition, atherapeutically effective amount of an inventive polypeptide, orpharmaceutically acceptable form thereof. Exemplary diseases, disorders,or conditions which may be treated by administration of an inventivepolypeptide comprise proliferative, neurological, immunological,endocrinologic, cardiovascular, hematologic, and inflammatory diseases,disorders, or conditions, and conditions characterized by premature orunwanted cell death.

As used herein a proliferative disease, condition, or disorder includes,but is not limited to, cancer, hematopoietic neoplastic disorders,proliferative breast disease, proliferative disorders of the lung,proliferative disorders of the colon, proliferative disorders of theliver, and proliferative disorders of the ovary.

Examples of cancers treatable by the above method include carcinoma,sarcoma, or metastatic disorders, breast cancer, ovarian cancer, coloncancer, lung cancer, fibrosarcoma, myosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer,pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer,cancer of the head and neck, skin cancer, brain cancer, squamous cellcarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcancer, testicular cancer, small cell lung carcinoma, non-small celllung carcinoma, bladder carcinoma, epithelial carcinoma, glioma,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposisarcoma,

Examples of hematopoietic neoplastic disorders treatable by the abovemethod includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. In certain embodiments, thediseases arise from poorly differentiated acute leukemias, e.g.,erythroblastic leukemia and acute megakaryoblastic leukemia. Additionalexemplary myeloid disorders include, but are not limited to, acutepromyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. inOncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stembergdisease.

Examples of proliferative breast disease treatable by the above methodincludes epithelial hyperplasia, sclerosing adenosis, and small ductpapillomas; tumors, e.g., stromal tumors such as fibroadenoma, phyllodestumor, and sarcomas, and epithelial tumors such as large duct papilloma;carcinoma of the breast including in situ (noninvasive) carcinoma thatincludes ductal carcinoma in situ (including Paget's disease) andlobular carcinoma in situ, and invasive (infiltrating) carcinomaincluding, but not limited to, invasive ductal carcinoma, invasivelobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma,tubular carcinoma, and invasive papillary carcinoma, and miscellaneousmalignant neoplasms. Disorders in the male breast include, but are notlimited to, gynecomastia and carcinoma.

Examples of proliferative disorders of the lung treatable by the abovemethod include, but are not limited to, bronchogenic carcinoma,including paraneoplastic syndromes, bronchioloalveolar carcinoma,neuroendocrine tumors, such as bronchial carcinoid, miscellaneoustumors, and metastatic tumors; pathologies of the pleura, includinginflammatory pleural effusions, noninflammatory pleural effusions,pneumothorax, and pleural tumors, including solitary fibrous tumors(pleural fibroma) and malignant mesothelioma.

Examples of proliferative disorders of the colon treatable by the abovemethod include, but are not limited to, non-neoplastic polyps, adenomas,familial syndromes, colorectal carcinogenesis, colorectal carcinoma, andcarcinoid tumors.

Examples of proliferative disorders of the liver treatable by the abovemethod include, but are not limited to, nodular hyperplasias, adenomas,and malignant tumors, including primary carcinoma of the liver andmetastatic tumors.

Examples of proliferative disorders of the ovary treatable by the abovemethod include, but are not limited to, ovarian tumors such as, tumorsof coelomic epithelium, serous tumors, mucinous tumors, endometeriodtumors, clear cell adenocarcinoma, cystadenofibroma, brenner tumor,surface epithelial tumors; germ cell tumors such as mature (benign)teratomas, monodermal teratomas, immature malignant teratomas,dysgerminoma, endodermal sinus tumor, choriocarcinoma; sex cord-stomaltumors such as, granulosa-theca cell tumors, thecomafibromas,androblastomas, hill cell tumors, and gonadoblastoma; and metastatictumors such as Krukenberg tumors.

The polypeptides described herein can also be used to treat, prevent ordiagnose conditions characterised by overactive cell death or cellulardeath due to physiologic insult etc. Some examples of conditionscharacterized by premature or unwanted cell death are or alternativelyunwanted or excessive cellular proliferation include, but are notlimited to hypocellular/hypoplastic, acellular/aplastic, orhypercellular/hyperplastic conditions. Some examples include hematologicdisorders including but not limited to fanconi anemia, aplastic anemia,thalaessemia, congenital neutropenia, myelodysplasia. The polypeptidesof the invention that act to decrease apoptosis can be used to treatdisorders associated with an undesirable level of cell death. Thus, theanti-apoptotic peptides of the invention can be used to treat disorderssuch as those that lead to cell death associated with viral infection,e.g., infection associated with infection with human immunodeficiencyvirus (HIV).

A wide variety of neurological diseases are characterized by the gradualloss of specific sets of neurons, and the anti-apoptotic peptides can beused in the treatment of these disorders. Such disorders includeAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis(ALS) retinitis pigmentosa, spinal muscular atrophy, and various formsof cerebellar degeneration. The cell loss in these diseases does notinduce an inflammatory response, and apoptosis appears to be themechanism of cell death. In addition, a number of hematologic diseasesare associated with a decreased production of blood cells. Thesedisorders include anemia associated with chronic disease, aplasticanemia, chronic neutropenia, and the myelodysplastic syndromes.Disorders of blood cell production, such as myelodysplastic syndrome andsome forms of aplastic anemia, are associated with increased apoptoticcell death within the bone marrow. These disorders could result from theactivation of genes that promote apoptosis, acquired deficiencies instromal cells or hematopoietic survival factors, or the direct effectsof toxins and mediators of immune responses. Two common disordersassociated with cell death are myocardial infarctions and stroke. Inboth disorders, cells within the central area of ischemia, which isproduced in the event of acute loss of blood flow, appear to die rapidlyas a result of necrosis. However, outside the central ischemic zone,cells die over a more protracted time period and morphologically appearto die by apoptosis. The anti-apoptotic peptides of the invention can beused to treat all such disorders associated with undesirable cell death.

Some examples of neurologic disorders that can be treated with thepolypeptides described herein include but are not limited to Alzheimer'sDisease, Down's Syndrome, Dutch Type Hereditary Cerebral HemorrhageAmyloidosis, Reactive Amyloidosis, Familial Amyloid Nephropathy withUrticaria and Deafness, Muckle-Wells Syndrome, Idiopathic Myeloma;Macroglobulinemia-Associated Myeloma, Familial Amyloid Polyneuropathy,Familial Amyloid Cardiomyopathy, Isolated Cardiac Amyloid, SystemicSenile Amyloidosis, Adult Onset Diabetes, Insulinoma, Isolated AtrialAmyloid, Medullary Carcinoma of the Thyroid, Familial Amyloidosis,Hereditary Cerebral Hemorrhage With Amyloidosis, Familial AmyloidoticPolyneuropathy, Scrapie, Creutzfeldt-Jacob Disease, GerstmannStraussler-Scheinker Syndrome, Bovine Spongiform Encephalitis, aPrion-mediated disease, Huntington's Disease, Pick's Disease,Amyotrophic Lateral Schlerosis (ALS), Parkinson's Disease, and Lewy BodyDisease.

Some examples of endocrinologic disorders that can be treated with thepolypeptides described herein include but are not limited to diabetes,hypothyroidism, hypopituitarism, hypoparathyroidism, hypogonadism,fertility disorders, etc.

Some examples of immunologic disorders that can be treated with thepolypeptides described herein include but are not limited to organtransplant rejection, arthritis, lupus, IBD, Crohn's disease, asthma,multiple sclerosis, diabetes, Graft versus host diseases, autoimmunediseases, psoriasis, rheumatoid arthritis, etc.

Examples of cardiovascular disorders that can be treated or preventedwith the the polypeptides of the invention include, but are not limitedto, atherosclerosis, myocardial infarction, stroke, thrombosis,aneurism, heart failure, ischemic heart disease, angina pectoris, suddencardiac death, hypertensive heart disease; non-coronary vessel disease,such as arteriolosclerosis, small vessel disease, nephropathy,hypertriglyceridemia, hypercholesterolernia, hyperlipidemia,xanthomatosis, asthma, hypertension, emphysema and chronic pulmonarydisease; or a cardiovascular condition associated with interventionalprocedures (“procedural vascular trauma”), such as restenosis followingangioplasty, placement of a shunt, stent, synthetic or natural excisiongrafts, indwelling catheter, valve or other implantable devices.

The inventive stitched polypeptides may serve to treat theabove-described diseases, disorders, or conditions, by disrupting nativeprotein-protein, protein-ligand, and/or protein-receptor interactions.For example, many biologically important protein/protein interactions,such as p53/MDM2 and Bcl-X1/Bak, are mediated by one protein donating ahelix into a cleft of its helix-accepting partner. The interaction ofp53 and MDM2 and mutations in the p53 gene have been identified invirtually half of all reported cancer cases (see, Shair Chem. & Biol.1997, 4, 791, the entire contents of which are incorporated herein byreference). As stresses are imposed on a cell, p53 is believed toorchestrate a response that leads to either cell-cycle arrest and DNArepair, or programmed cell death. As well as mutations in the p53 genethat alter the function of the p53 protein directly, p53 can be alteredby changes in MDM2. The MDM2 protein has been shown to bind to p53 anddisrupt transcriptional activation by associating with thetransactivation domain of p53. For example, an 11 amino-acid peptidederived from the transactivation domain of p53 forms an amphipathicalpha-helix of 2.5 turns that inserts into the MDM2 crevice.

Thus, in certain embodiments, an inventive polypeptide is an alphahelical polypeptide that is capable of binding tightly to a helixacceptor and disrupting native protein/protein interactions. Thesestructures may then be screened using high throughput techniques toidentify optimal small molecule peptides. In certain embodiments, aninventive polypeptide is an alpha helical p53 polypeptide capable ofbinding to the Xenopus MDM2 protein. The novel structures that disruptthe MDM2 interaction might be useful for many applications, including,but not limited to, control of soft tissue sarcomas (which overexpressesMDM2 in the presence of wild type p53). These cancers may be held incheck with small molecules that could intercept MDM2, thereby preventingsuppression of p53. Additionally, small molecules disrupters of MDM2-p53interactions could be used as adjuvant therapy to help control andmodulate the extent of the p53 dependent apoptosis response inconventional chemotherapy.

In certain embodiments, the inventive polypeptide is homologous to aknown alpha helical peptide. In certain embodiments, the inventivepolypeptide is at least 80%, 85%, 90%, or 95% homologous to a knownalpha helical peptide.

In addition, the inventive polypeptides may be useful in the area ofmaterials science. For example, molecules such as lipids and otherpolymeric molecules may be attached to the terminal peptide moieties andthus generate potentially important biomaterials.

In addition to the above-mentioned uses, the inventive polypeptides maybe used for studies in bioinorganic chemistry or in catalysis, either asa ligand for a transition metal capable of mimicking an importantbiological environment, or by acting in concert with a particulartransition metal catalyst to effect a desired chemical reaction.

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions comprising aninventive stitched polypeptide, or pharmaceutically acceptable formthereof, and a pharmaceutically acceptable carrier. Such pharmaceuticalcompositions may optionally comprise one or more additionalbiologically-active substances. In accordance with some embodiments, amethod of administering a pharmaceutical composition comprisinginventive compositions to a subject in need thereof is provided. In someembodiments, inventive compositions are administered to humans. For thepurposes of the present invention, the phrase “active ingredient”generally refers to an inventive polypeptide, as described herein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and/or other primates; mammals, includingcommercially relevant mammals such as cattle, pigs, horses, sheep, cats,and/or dogs; and/or birds, including commercially relevant birds such aschickens, ducks, geese, and/or turkeys.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, shaping and/or packaging the product into a desired single-or multi-dose unit.

A pharmaceutical composition of the invention may be prepared, packaged,and/or sold in bulk, as a single unit dose, and/or as a plurality ofsingle unit doses. As used herein, a “unit dose” is discrete amount ofthe pharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject and/or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and/or any additional ingredients in apharmaceutical composition of the invention will vary, depending uponthe identity, size, and/or condition of the subject treated and furtherdepending upon the route by which the composition is to be administered.By way of example, the composition may comprise between 0.1% and 100%(w/w) active ingredient.

Pharmaceutical formulations of the present invention may additionallycomprise a pharmaceutically acceptable excipient, which, as used herein,includes any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, Md.,2006) discloses various carriers used in formulating pharmaceuticalcompositions and known techniques for the preparation thereof. Exceptinsofar as any conventional carrier medium is incompatible with asubstance or its derivatives, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition, its use iscontemplated to be within the scope of this invention.

In some embodiments, the pharmaceutically acceptable excipient is atleast 95%, 96%, 97%, 98%, 99%, or 100% pure. In some embodiments, theexcipient is approved for use in humans and for veterinary use. In someembodiments, the excipient is approved by United States Food and DrugAdministration. In some embodiments, the excipient is pharmaceuticalgrade. In some embodiments, the excipient meets the standards of theUnited States Pharmacopoeia (USP), the European Pharmacopoeia (EP), theBritish Pharmacopoeia, and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in the inventive formulations.Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and combinations thereof

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds,etc., and combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminumsilicate]), long chain amino acid derivatives, high molecular weightalcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetinmonostearate, ethylene glycol distearate, glyceryl monostearate, andpropylene glycol monostearate, polyvinyl alcohol), carbomers (e.g.carboxy polymethylene, polyacrylic acid, acrylic acid polymer, andcarboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60],polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate[Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span65], glyceryl monooleate, sorbitan monooleate [Span 80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj 45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and Solutol), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [Brij 30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural andsynthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larcharabogalactan); alginates; polyethylene oxide; polyethylene glycol;inorganic calcium salts; silicic acid; polymethacrylates; waxes; water;alcohol; etc.; and combinations thereof.

Exemplary preservatives may include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, alcoholpreservatives, acidic preservatives, and other preservatives. Exemplaryantioxidants include, but are not limited to, alpha tocopherol, ascorbicacid, acorbyl palmitate, butylated hydroxyanisole, butylatedhydroxytoluene, monothioglycerol, potassium metabisulfite, propionicacid, propyl gallate, sodium ascorbate, sodium bisulfite, sodiummetabisulfite, and sodium sulfite. Exemplary chelating agents includeethylenediaminetetraacetic acid (EDTA), citric acid monohydrate,disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malicacid, phosphoric acid, sodium edetate, tartaric acid, and trisodiumedetate. Exemplary antimicrobial preservatives include, but are notlimited to, benzalkonium chloride, benzethonium chloride, benzylalcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine,chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol,glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethylalcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.Exemplary antifungal preservatives include, but are not limited to,butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoicacid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodiumbenzoate, sodium propionate, and sorbic acid. Exemplary alcoholpreservatives include, but are not limited to, ethanol, polyethyleneglycol, phenol, phenolic compounds, bisphenol, chlorobutanol,hydroxybenzoate, and phenylethyl alcohol. Exemplary acidic preservativesinclude, but are not limited to, vitamin A, vitamin C, vitamin E,beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbicacid, sorbic acid, and phytic acid. Other preservatives include, but arenot limited to, tocopherol, tocopherol acetate, deteroxime mesylate,cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened(BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ethersulfate (SLES), sodium bisulfite, sodium metabisulfite, potassiumsulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben,Germall 115, Germaben II, Neolone, Kathon, and Euxyl. In certainembodiments, the preservative is an anti-oxidant. In other embodiments,the preservative is a chelating agent.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., andcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and combinations thereof.

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredients, the liquid dosage forms may comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, the oral compositions caninclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, and perfuming agents. In certainembodiments for parenteral administration, the conjugates of theinvention are mixed with solubilizing agents such as Cremophor,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may be a sterile injectable solution,suspension or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing the conjugates of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may comprise buffering agents.

Solid compositions of a similar type may be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The active ingredients can be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active ingredient may be admixed with at least oneinert diluent such as sucrose, lactose or starch. Such dosage forms maycomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may comprise bufferingagents. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of aconjugate of this invention may include ointments, pastes, creams,lotions, gels, powders, solutions, sprays, inhalants and/or patches.Generally, the active component is admixed under sterile conditions witha pharmaceutically acceptable carrier and/or any needed preservativesand/or buffers as may be required. Additionally, the present inventioncontemplates the use of transdermal patches, which often have the addedadvantage of providing controlled delivery of an active ingredient tothe body. Such dosage forms may be prepared, for example, by dissolvingand/or dispensing the active ingredient in the proper medium.Alternatively or additionally, the rate may be controlled by eitherproviding a rate controlling membrane and/or by dispersing the activeingredient in a polymer matrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositionsmay be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 and functional equivalents thereof. Jetinjection devices which deliver liquid vaccines to the dermis via aliquid jet injector and/or via a needle which pierces the stratumcorneum and produces a jet which reaches the dermis are suitable. Jetinjection devices are described, for example, in U.S. Pat. Nos.5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis are suitable. Alternatively or additionally, conventionalsyringes may be used in the classical mantoux method of intradermaladministration.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,and/or sold in a formulation suitable for pulmonary administration viathe buccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers or from about 1 to about 6nanometers. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder and/or using a self propelling solvent/powder dispensingcontainer such as a device comprising the active ingredient dissolvedand/or suspended in a low-boiling propellant in a sealed container. Suchpowders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositions mayinclude a solid fine powder diluent such as sugar and are convenientlyprovided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic and/or solid anionic surfactant and/or a solid diluent(which may have a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may provide the active ingredient in the form of droplets of asolution and/or suspension. Such formulations may be prepared, packaged,and/or sold as aqueous and/or dilute alcoholic solutions and/orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization and/oratomization device. Such formulations may further comprise one or moreadditional ingredients including, but not limited to, a flavoring agentsuch as saccharin sodium, a volatile oil, a buffering agent, a surfaceactive agent, and/or a preservative such as methylhydroxybenzoate. Thedroplets provided by this route of administration may have an averagediameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare useful for intranasal delivery of a pharmaceutical composition ofthe invention. Another formulation suitable for intranasaladministration is a coarse powder comprising the active ingredient andhaving an average particle from about 0.2 to 500 micrometers. Such aformulation is administered in the manner in which snuff is taken, i.e.by rapid inhalation through the nasal passage from a container of thepowder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition of theinvention may be prepared, packaged, and/or sold in a formulationsuitable for buccal administration. Such formulations may, for example,be in the form of tablets and/or lozenges made using conventionalmethods, and may, for example, 0.1 to 20% (w/w) active ingredient, thebalance comprising an orally dissolvable and/or degradable compositionand, optionally, one or more of the additional ingredients describedherein. Alternately, formulations suitable for buccal administration maycomprise a powder and/or an aerosolized and/or atomized solution and/orsuspension comprising the active ingredient. Such powdered, aerosolized,and/or aerosolized formulations, when dispersed, may have an averageparticle and/or droplet size in the range from about 0.1 to about 200nanometers, and may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,and/or sold in a formulation suitable for ophthalmic administration.Such formulations may, for example, be in the form of eye dropsincluding, for example, a 0.1/1.0% (w/w) solution and/or suspension ofthe active ingredient in an aqueous or oily liquid carrier. Such dropsmay further comprise buffering agents, salts, and/or one or more otherof the additional ingredients described herein. Otheropthalmically-administrable formulations which are useful include thosewhich comprise the active ingredient in microcrystalline form and/or ina liposomal preparation. Ear drops and/or eye drops are contemplated asbeing within the scope of this invention.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st)ed., Lippincott Williams &Wilkins, 2005.

Administration

In some embodiments, a therapeutically effective amount of an inventivepharmaceutical composition is delivered to a patient and/or organismprior to, simultaneously with, and/or after diagnosis with a disease,disorder, and/or condition. In some embodiments, a therapeutic amount ofan inventive composition is delivered to a patient and/or organism priorto, simultaneously with, and/or after onset of symptoms of a disease,disorder, and/or condition. In some embodiments, the amount of inventiveconjugate is sufficient to treat, alleviate, ameliorate, relieve, delayonset of, inhibit progression of, reduce severity of, and/or reduceincidence of one or more symptoms or features of the disease, disorder,and/or condition.

The compositions, according to the method of the present invention, maybe administered using any amount and any route of administrationeffective for treatment. The exact amount required will vary fromsubject to subject, depending on the species, age, and general conditionof the subject, the severity of the infection, the particularcomposition, its mode of administration, its mode of activity, and thelike. The compositions of the invention are typically formulated indosage unit form for ease of administration and uniformity of dosage. Itwill be understood, however, that the total daily usage of thecompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective dose level for any particular subject ororganism will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; the activity of thespecific active ingredient employed; the specific composition employed;the age, body weight, general health, sex and diet of the subject; thetime of administration, route of administration, and rate of excretionof the specific active ingredient employed; the duration of thetreatment; drugs used in combination or coincidental with the specificactive ingredient employed; and like factors well known in the medicalarts.

The pharmaceutical compositions of the present invention may beadministered by any route. In some embodiments, the pharmaceuticalcompositions of the present invention are administered variety ofroutes, including oral, intravenous, intramuscular, intra-arterial,intramedullary, intrathecal, subcutaneous, intraventricular,transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical(as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal,enteral, sublingual; by intratracheal instillation, bronchialinstillation, and/or inhalation; and/or as an oral spray, nasal spray,and/or aerosol. Specifically contemplated routes are systemicintravenous injection, regional administration via blood and/or lymphsupply, and/or direct administration to an affected site. In general themost appropriate route of administration will depend upon a variety offactors including the nature of the agent (e.g., its stability in theenvironment of the gastrointestinal tract), the condition of the subject(e.g., whether the subject is able to tolerate oral administration),etc. At present the oral and/or nasal spray and/or aerosol route is mostcommonly used to deliver therapeutic agents directly to the lungs and/orrespiratory system. However, the invention encompasses the delivery ofthe inventive pharmaceutical composition by any appropriate route takinginto consideration likely advances in the sciences of drug delivery.

In certain embodiments, the conjugates of the invention may beadministered at dosage levels sufficient to deliver from about 0.001mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, fromabout 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg toabout 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject bodyweight per day, one or more times a day, to obtain the desiredtherapeutic effect. The desired dosage may be delivered three times aday, two times a day, once a day, every other day, every third day,every week, every two weeks, every three weeks, or every four weeks. Incertain embodiments, the desired dosage may be delivered using multipleadministrations (e.g., two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, or more administrations).

In some embodiments, the present invention encompasses “therapeuticcocktails” comprising inventive polypeptides. In some embodiments, theinventive polypeptide comprises a single species which can bind tomultiple targets. In some embodiments, different inventive polypeptidescomprise different targeting moiety species, and all of the differenttargeting moiety species can bind to the same target. In someembodiments, different inventive polypeptides comprise differenttargeting moiety species, and all of the different targeting moietyspecies can bind to different targets. In some embodiments, suchdifferent targets may be associated with the same cell type. In someembodiments, such different targets may be associated with differentcell types.

It will be appreciated that inventive polypeptides and pharmaceuticalcompositions of the present invention can be employed in combinationtherapies. The particular combination of therapies (therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will be appreciated thatthe therapies employed may achieve a desired effect for the same purpose(for example, an inventive conjugate useful for detecting tumors may beadministered concurrently with another agent useful for detectingtumors), or they may achieve different effects (e.g., control of anyadverse effects).

Pharmaceutical compositions of the present invention may be administeredeither alone or in combination with one or more other therapeuticagents. By “in combination with,” it is not intended to imply that theagents must be administered at the same time and/or formulated fordelivery together, although these methods of delivery are within thescope of the invention. The compositions can be administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutics or medical procedures. In general, each agent will beadministered at a dose and/or on a time schedule determined for thatagent. Additionally, the invention encompasses the delivery of theinventive pharmaceutical compositions in combination with agents thatmay improve their bioavailability, reduce and/or modify theirmetabolism, inhibit their excretion, and/or modify their distributionwithin the body.

The particular combination of therapies (therapeutics and/or procedures)to employ in a combination regimen will take into account compatibilityof the desired therapeutics and/or procedures and/or the desiredtherapeutic effect to be achieved. It will be appreciated that thetherapies employed may achieve a desired effect for the same disorder(for example, an inventive polypeptide may be administered concurrentlywith another biologically active agent used to treat the same disorder),and/or they may achieve different effects (e.g., control of any adverseeffects). In some embodiments, polypeptides of the invention areadministered with a second biologically active agent that is approved bythe U.S. Food and Drug Administration.

In will further be appreciated that biologically active agents utilizedin this combination may be administered together in a single compositionor administered separately in different compositions.

In general, it is expected that biologically active agents utilized incombination be utilized at levels that do not exceed the levels at whichthey are utilized individually. In some embodiments, the levels utilizedin combination will be lower than those utilized individually.

In some embodiments, inventive pharmaceutical compositions may beadministered in combination with any biologically active agent ortherapeutic regimen that is useful to treat, alleviate, ameliorate,relieve, delay onset of, inhibit progression of, reduce severity of,and/or reduce incidence of one or more symptoms or features of cancer.For example, inventive compositions may be administered in combinationwith traditional cancer therapies including, but not limited to,surgery, chemotherapy, radiation therapy, hormonal therapy,immunotherapy, complementary or alternative therapy, and any combinationof these therapies.

In some embodiments, inventive compositions are administered incombination with surgery to remove a tumor. Because complete removal ofa tumor with minimal or no damage to the rest of a patient's body istypically the goal of cancer treatment, surgery is often performed tophysically remove part or all of a tumor. If surgery is unable tocompletely remove a tumor, additional therapies (e.g. chemotherapy,radiation therapy, hormonal therapy, immunotherapy, complementary oralternative therapy) may be employed.

In some embodiments, inventive compositions are administered incombination with radiation therapy. Radiation therapy (also known asradiotherapy, X-ray therapy, or irradiation) is the use of ionizingradiation to kill cancer cells and shrink tumors. Radiation therapy maybe used to treat almost any type of solid tumor, including cancers ofthe brain, breast, cervix, larynx, lung, pancreas, prostate, skin,stomach, uterus, or soft tissue sarcomas. Radiation can be used to treatleukemia and lymphoma. Radiation therapy can be administered externallyvia external beam radiotherapy (EBRT) or internally via brachytherapy.Typically, the effects of radiation therapy are localized and confinedto the region being treated. Radiation therapy injures or destroys tumorcells in an area being treated (e.g. a target organ, tissue, and/orcell) by damaging their genetic material, preventing tumor cells fromgrowing and dividing. In general, radiation therapy attempts to damageas many tumor cells as possible while limiting harm to nearby healthytissue. Hence, it is often administered in multiple doses, allowinghealthy tissue to recover between fractions.

In some embodiments, inventive compositions are administered incombination with immunotherapy. Immunotherapy is the use of immunemechanisms against tumors which can be used in various forms of cancer,such as breast cancer (e.g. trastuzumab/Herceptin®), leukemia (e.g.gemtuzumab ozogamicin/Mylotarg®), and non-Hodgkin's lymphoma (e.g.rituximab/Rituxan®). In some embodiments, immunotherapy agents aremonoclonal antibodies directed against proteins that are characteristicto the cells of the cancer in question. In some embodiments,immunotherapy agents are cytokines that modulate the immune system'sresponse. In some embodiments, immunotherapy agents may be vaccines.

In some embodiments, vaccines can be administered to prevent and/ordelay the onset of cancer. In some embodiments, cancer vaccines preventand/or delay the onset of cancer by preventing infection by oncogenicinfectious agents. In some embodiments, cancer vaccines prevent and/ordelay the onset of cancer by mounting an immune response againstcancer-specific epitopes. To give but one example of a cancer vaccine,an experimental vaccine for HPV types 16 and 18 was shown to be 100%successful at preventing infection with these types of HPV and, thus,are able to prevent the majority of cervical cancer cases (Harper etal., 2004, Lancet, 364:1757).

In some embodiments, inventive compositions are administered incombination with complementary and alternative medicine treatments. Someexemplary complementary measures include, but are not limited to,botanical medicine (e.g. use of mistletoe extract combined withtraditional chemotherapy for the treatment of solid tumors); acupuncturefor managing chemotherapy-associated nausea and vomiting and incontrolling pain associated with surgery; prayer; psychologicalapproaches (e.g. “imaging” or meditation) to aid in pain relief orimprove mood. Some exemplary alternative measures include, but are notlimited to, diet and other lifestyle changes (e.g. plant-based diet, thegrape diet, and the cabbage diet).

In some embodiments, inventive compositions are administered incombination with any of the traditional cancer treatments describedherein, which are often associated with unpleasant, uncomfortable,and/or dangerous side effects. For example, chronic pain often resultsfrom continued tissue damage due to the cancer itself or due to thetreatment (i.e., surgery, radiation, chemotherapy). Alternatively oradditionally, such therapies are often associated with hair loss,nausea, vomiting, diarrhea, constipation, anemia, malnutrition,depression of immune system, infection, sepsis, hemorrhage, secondaryneoplasms, cardiotoxicity, hepatotoxicity, nephrotoxicity, ototoxicity,etc. Thus, inventive compositions which are administered in combinationwith any of the traditional cancer treatments described herein may bealso be administered in combination with any therapeutic agent ortherapeutic regimen that is useful to treat, alleviate, ameliorate,relieve, delay onset of, inhibit progression of, reduce severity of,and/or reduce incidence of one or more side effects of cancer treatment.To give but a few examples, pain can be treated with opioids and/oranalgesics (e.g. morphine, oxycodone, antiemetics, etc.); nausea andvomiting can be treated with 5-HT₃ inhibitors (e.g. dolasetron/Anzemet®,granisetron/Kytril®, ondansetron/Zofran®, palonsetron/Aloxi®) and/orsubstance P inhibitors (e.g. aprepitant/Emend®); immunosuppression canbe treated with a blood transfusion; infection and/or sepsis can betreated with antibiotics (e.g. penicillins, tetracyclines,cephalosporins, sulfonamides, aminoglycosides, etc.); and so forth.

In some embodiments, inventive compositions may be administered and/orinventive diagnostic methods may be performed in combination with anytherapeutic agent or therapeutic regimen that is useful to diagnose oneor more symptoms or features of cancer (e.g. detect the presence ofand/or locate a tumor). In some embodiments, inventive conjugates may beused in combination with one or more other diagnostic agents. To givebut one example, conjugates used to detect tumors may be administered incombination with other agents useful in the detection of tumors. Forexample, inventive conjugates may be administered in combination withtraditional tissue biopsy followed by immunohistochemical staining andserological tests (e.g. prostate serum antigen test). Alternatively oradditionally, inventive conjugates may be administered in combinationwith a contrasting agent for use in computed tomography (CT) scansand/or MRI.

Kits

The invention provides a variety of kits comprising one or more of thepolypeptides of the invention. For example, the invention provides a kitcomprising an inventive polypeptide and instructions for use. A kit maycomprise multiple different polypeptides. A kit may comprise any of anumber of additional components or reagents in any combination. All ofthe various combinations are not set forth explicitly but eachcombination is included in the scope of the invention.

According to certain embodiments of the invention, a kit may include,for example, (i) one or more inventive polypeptides and one or moreparticular biologically active agents to be delivered; (ii) instructionsfor administering the conjugate to a subject in need thereof.

Kits typically include instructions which may, for example, compriseprotocols and/or describe conditions for production of inventivepolypeptides, administration of inventive polypeptides to a subject inneed thereof, design of novel inventive polypeptides, etc. Kits willgenerally include one or more vessels or containers so that some or allof the individual components and reagents may be separately housed. Kitsmay also include a means for enclosing individual containers inrelatively close confinement for commercial sale, e.g., a plastic box,in which instructions, packaging materials such as styrofoam, etc., maybe enclosed. An identifier, e.g., a bar code, radio frequencyidentification (ID) tag, etc., may be present in or on the kit or in orone or more of the vessels or containers included in the kit. Anidentifier can be used, e.g., to uniquely identify the kit for purposesof quality control, inventory control, tracking, movement betweenworkstations, etc.

EXEMPLIFICATION

The present invention will be more specifically illustrated by thefollowing examples. However, it should be understood that the presentinvention is not limited by these examples in any manner.

Example 1. Stitching Alpha-Helical Peptides by Tandem Ring-ClosingMetathesis

For the bis-olefinic amino acid that provides the spiro junction of thestitched peptide, we chose bis-pentenylglycine (B₅) (FIG. 1D). Studieswith single hydrocarbon staples had established that five-carbon chainlength in B₅ to be optimal at the C-terminal end of the i,i+4 staple,when S-configurated and combined with an N-terminal S₅ residue; and atthe N-terminal end of the i,i+7 staple, when R-configurated and combinedwith a C-terminal S₈ residue. (Schafmeister et al. J. Am. Chem. Soc.(2000) 122:5891-5892). Peptides containing an N-terminal S₅ (i), centralB₅ (i+4) and C-terminal S₈ (i+4+7) bear four terminal olefins, which areequivalent electronically but differentiated regiochemically by virtueof their attachment to the peptide framework.

Considering only intramolecular reaction pathways, tandem-RCM couldproduce three regioisomeric products, 2, 3 and 4 (FIG. 1A). Ofparticular concern was the possibility that the two olefins in B₅ mightpreferentially react with each other during RCM (reaction a), becausethe resulting 9-membered ring would be smaller than either of thoseproduced by inter-residue RCM.

To investigate all the possible reaction pathways, we turned to modelstudies examining each in isolation using the sequence of the C-peptideof RNase A (Bierzynski, A.; Kim, P. S.; Baldwin, R. L. Proc. Acad. Sci.U.S.A. 1982, 79, 2470-2474). A model peptide designed to test reaction aby incorporating only B₅, was a poor substrate for RCM (Table 5, entryII), probably owing to ring strain in the transition state leading tothe cyclononenyl product A literature search failed to produce anyreported example of RCM leading to cyclononenyl product. The ethyl esterof Fmoc amino acid B₅ also failed to form the cyclononenyl product undersimilar conditions; instead, a dimeric 18-membered metathesis productwas formed as the exclusive product (Scheme 2).

TABLE 5 Sequences of Peptide Substrates andPercent Conversions for Metathesis Reaction. % SEQ Rxn conversion^(b)Substrate sequence^(a) ID No. modeled 2h +2h^(c) I Ac-EWAETAAAKFLAAHA, 9SEQ ID 1 — — II Ac-EWAETAAB ₅KFLAAHA SEQ ID 2 a <2^(d) <2^(d) III Ac-EWA

TAAAKFLAAH

SEQ ID 3 b <2^(d) <2^(d) IV Ac-EWA

TAA

KFLAAHA SEQ ID 4 c <2^(d) <2^(d) V Ac-EWAETAA

KFLAAH

SEQ ID 5 d  48 — VI Ac-EWA

TAA

KFLAAHA SEQ ID 6 e >98 — VII Ac-EWAETAA

KFLAAH

SEQ ID 7 f >98 — VIII Ac-EWAS ₅TAA

KFLAAH*^(e) SEQ ID 8 (product 6) —  98 IX Ac-EWA*TAA

KFLAAH

^(e) SEQ ID 9 (product 5) — >98 X Ac-EWA

TAAB ₅KFLAAH

SEQ ID 10 (product 4^(f)) — >98 XI Ac-EWA

TAAB ₅KFL

AHA SEQ ID 11 (product 8^(f)) — >98 ^(a)Metathesis was performed onsolid support with the fully protected peptide using 20 mol% Grubbscatalyst^(4b) in dichloroethane. ^(b)Percent conversion[product/(product + starting material)] as determined by reversed-phaseHPLC following cleavage from resin. ^(c)Product yield following a second2-hour metathesis reaction using fresh catalyst. ^(d)RCM product was notdetected. ^(e)Asterisk represents alpha-aminoisobutyric acid (Aib),which was incorporated to mimic the helix-stabilizing effect of thealpha, alpha-disubstituted amino acids S5 and Sg. ^(f)Double RCMproduct.

A peptide configured to test reaction b also failed to yield appreciableamounts of product (entry III, Table 5). These results having thusindicated that the a+b tandem-RCM pathway is disfavored, the tworemaining alternatives were c+d and e+f. In model peptides, reaction cfailed and d gave only modest yields (entries IV and V, respectively).On the other hand, both reactions e and f proceeded efficiently (entriesVI and VII, respectively), as expected from previous studies (seeSchafmeister, C. E.; Po, J.; Verdine, G. L. J. Am. Chem. Soc. 2000, 122,5891-5892). The exquisite selectivity of RCM in these peptides isclearly evident from comparison of entry VI with IV, in which inversionof a single stereogenic center causes a nearly quantitative reaction tofail.

Of the six mono-RCM reactions, by far the two most efficient ones were eand f. Should this preferential reactivity be retained with a peptidecontaining all four olefinic tethers required to introduce a stitchedhelix, then the e+f pathway might be favored enough to provide product 4cleanly. To test this, we synthesized peptide 1 and subjected it to RCMunder the same conditions as used in the component reactions, thendeprotected the peptide and analyzed the products by LCMS. A singleproduct peak accounted for 90% of the product mixture, with theremainder being unreacted starting material. This product had themolecular mass expected of the product of tandem metathesis (i.e., 1minus 2 mol equivalents of ethylene). Edman degradation revealed thatonly the olefin-containing amino acids had been altered in the RCMreaction. By subjecting resin-bound 1 to a second round of RCM, we wereable to increase the product conversion to greater than 98%. The resultsof the mono-RCM reactions had suggested 4 to be the most likelystructure for the tandem-RCM product, and this assignment was confirmedby computational analysis of the two possible stitched products, 3 and4; Molecular modeling indicated that the lowest energy double bondisomer of product 4 is lower in energy than the most stable isomer of 3by ˜15 kcal/mol. This is in part due to three syn-pentane interactionsthat arise in product 3. Computational analysis further indicated a ˜2.5kcal/mol preference for the i,i+4 olefin to be configurated cis; thei,i+7 olefin has no such configurational bias, and therefore theintrinsic preference of the catalyst to produce trans olefins probablydominates.

Circular dichroism (CD) measurements were performed to determine theeffects of stitching on the conformational preferences and thermalstability of the peptides. Stitched peptide 4 displayed thecharacteristic CD signature of alpha-helices, but was less affected byincreasing temperature than single-stapled peptides 5 and 6 (FIGS. 2A,2B, and 3B). Indeed, whereas 5 underwent a cooperative meltingtransition at 57° C., 4 retained more than 50% of its alpha-helicityeven at 95° C. (see FIG. 4 for additional melting data). The greaterhelix stability of peptide 4 than 5 was accompanied by enhancedresistance to tryptic digestion; even in the presence of a vast molarexcess of trypsin, the stitched peptide 4 exhibited a half-life ofnearly three hours (172 min, FIG. 2C).

To investigate the possibility of forming stitched peptides having thei+4+4 constitution, we again applied the half-site rules to designpeptide 7 (Table 1, entry XI). This substrate also underwent efficientRCM leading to a doubly crosslinked product. Computational analysisindicated that both olefins in the stitched product 8 (FIG. 1C) wouldhave to be cis-configurated in order to form a stable alpha-helix.Though 8 clearly exhibited helical character greater than the stapledpeptide 5 and less than that of the i+4+7 stitched peptide 4, theapparently complex melting behavior of 8 precluded accurate T_(m)determination.

Experiment General

Commercially available solvents and reagents were used as receivedunless otherwise indicated. Tetrahydrofuran (THF) was distilled fromsodium metal in the presence of benzophenone under dry nitrogen.Dichloromethane (CH₂Cl₂) was distilled from calcium hydride under drynitrogen. Reactions involving moisture-sensitive reagents were carriedout under an inert atmosphere of dry argon. All glassware was driedprior to use, and all liquid transfers were performed using dry syringesand needles. All NMR spectra were recorded on a Varian Mercury 400 modelspectrometer. Chemical shifts (δ) for ¹H and ¹³C NMR spectra arereported in ppm relative to residual solvent protons or carbons,respectively. High resolution ESI mass spectra were obtained using a LCTmass spectrometer (Micromass Inc., Beverly, Mass.). Peptides werepurified by reverse-phase HPLC with a 9.4×250 mm Agilent C₁₈ reversephase column using an Agilent 1100 series HPLC. Analysis of the purifiedpeptides was performed on an Agilent 1100 series LC/MSD electrospraytrap with a 3.5×150 mm Agilent C₁₈ reverse phase column.

Ethyl 2-(diphenylmethyleneamino)-2-(pent-4-enyl)hept-6-enoate (11)

A procedure previously described for dialkylation ofN-(diphenylmethylene)glycine ethyl ester 10 was used after modifications(see Denmark, S. E.; Stavenger, R. A.; Faucher, A.-M.; Edwards, J. P. J.Org. Chem. 1997, 62, 3375-3389): To a stirred solution ofN-(diphenylmethylene)glycine ethyl ester 10 (13.63 g, 51 mmol) in THF(250 mL) was added a solution of KHMDS (11.2 g, 56.1 mmol, 1.1 equiv.)in THF (56 mL) via a cannula at −78° C. over 15 min. After stirring at−78° C. for 1 h, the resulting orange-colored solution was treated with5-iodo-1-pentene (12 g, 61.2 mmol, 1.2 equiv.). The reaction mixture wasallowed to warm to room temperature and stirred for 2 h. The resultingsuspension was cooled to −40° C. and another solution of KHMDS (15.3 g,76.5 mmol, 1.5 equiv.) in THF (77 mL) was added via a cannula over 15min and stirred for 1 h. 5-iodo-1-pentene (16 g, 81.6 mmol, 1.6 equiv.)was then quickly added to the burgundy-colored mixture, and the reactionwas left to warm to room temperature overnight (16 h). The reaction wasquenched by addition of saturated NH₄Cl solution in water (100 mL). Theorganics were extracted with ethyl acetate (2×150 mL), washed withNa₂S₂O₃ solution and then with brine. The organic layer was dried overMgSO₄, filtered, and concentrated under reduced pressure. The resultingresidue was dried in vacuo overnight and used for the next reactionwithout further purification: ¹H-NMR (400 MHz, CDCl₃) δ 7.83-7.12 (m,10H), 5.80 (m, 2H), 5.02 (dd, J=17.2, 1.6 Hz, 2H), 4.96 (dd, J=10.4, 1.6Hz, 2H), 3.74 (q, J=6.8 Hz, 2H), 2.05 (dd, J=14.0, 7.2 Hz, 4H), 1.92 (m,4H), 1.45 (m, 4H), 1.13 (t, J=6.8 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ174.8, 166.0, 141.3, 138.9, 128.5, 128.2, 127.9, 115.0, 69.2, 60.5,37.5, 34.4, 23.3, 14.2; HRMS (ESI) m/z for C₂₇H₃₄NO₂ [M+H]⁺ calcd404.2589. found 404.2577.

Ethyl2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-(pent-4-enyl)hept-6-enoate(12)

To a stirred solution of crude ethyl2-(diphenylmethyleneamino)-2-(pent-4-enyl)hept-6-enoate 11 (18.2 g, 45.1mmol) in ethyl ether (200 mL) was added a 6N solution of hydrochloricacid (45 mL) at 0° C. over 45 min and the resulting mixture was stirredfor another 15 min. The organics were extracted in ethyl ether (2×100mL), and the combined etherial layer was concentrated. The residue wasdissolved in acetone (75 mL), to which a solution ofN-(9-fluorenylmethoxycarbonyloxy)succinimide (16 g, 47.5 mmol, 1.05equiv.) in acetone (75 mL) and a solution of sodium carbonate (19.1 g,180.4 mmol, 4.0 equiv.) in water (150 mL) were consecutively added. Theresulting mixture was stirred at room temperature for 16 h. The productwas extracted with ethyl acetate (2×150 mL) and the combined organiclayer was dried over MgSO₄, filtered, and concentrated under reducedpressure. The resulting residue was purified by silica gel columnchromatography (eluting with 7% ethyl acetate in n-hexanes) to give 12as a white solid: ¹H-NMR (400 MHz, CDCl₃) δ 7.77 (d, J=7.2 Hz, 2H), 7.62(d, J=8.0 Hz, 2H), 7.40 (t, J=7.2 Hz, 2H), 7.32 (dt, J=7.2, 0.8 Hz, 2H),5.90 (br s, 1H), 5.75 (m, 2H), 4.99 (d, J=17.6 Hz, 2H), 4.95 (d, J=11.2Hz, 2H), 4.39 (d, J=6.8 Hz, 2H), 4.25 (m, 3H), 2.35 (dt, J=12.8, 4.0 Hz,2H), 2.02 (m, 4H), 1.76 (dt, J=12.8, 4.0 Hz, 2H) 1.39 (m, 2H), 1.30 (t,J=7.2 Hz, 3H), 1.06 (m, 2H); ¹³C-NMR (100 MHz, CDCl₃) δ 174.2, 154.0,144.2, 141.6, 138.5, 127.9, 127.3, 125.3, 120.2, 115.1, 66.3, 64.2,62.1, 47.6, 35.3, 33.6, 23.6, 14.5; HRMS (ESI) m/z for C₂₉H₃₆NO₄ [M+H]⁺calcd 462.2644. found 462.2637.

2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-2-(pent-4-enyl)hept-6-enoicacid (B₅)

A procedure previously described for dealkylation of esters was usedafter modifications (see Node et al., J. Org. Chem. 1981, 46, 1991): Toa stirred solution of aluminum bromide (22.4 g, 84.0 mmol, 3.0 equiv.)in methyl sulfide (90 mL) was slowly added a solution of ethyl2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-(pent-4-enyl)hept-6-enoate12 (12.7 g, 27.5 mmol) in dichloromethane (90 mL) at 0° C. over 15 min.The resulting mixture was allowed to warm to room temperature andstirred for 24 h. The reaction mixture was poured into water andacidified with a diluted HCl. The product was extracted withdichloromethane (2×100 mL) and the combined organic layer was washedwith brine, dried over MgSO₄, and concentrated under reduced pressure.The residual yellowish solid was purified by silica gel columnchromatography (eluting with 7% methanol in dichloromethane) to give B₅as a white solid: ¹H-NMR (400 MHz, CDCl₃) δ 9.94 (bs, 1H), 7.78 (d,J=7.6 Hz, 2H), 7.61 (d, J=7.6 Hz, 2H), 7.41 (t, J=7.6 Hz, 2H), 7.33 (dt,J=7.6, 0.8 Hz, 2H), 5.75 (m, 2H), 5.00 (d, J=18.8 Hz, 2H), 4.96 (d,J=11.6 Hz, 2H), 4.42 (d, J=6.8 Hz, 2H), 4.23 (t, J=6.8 Hz, 1H), 2.34(dt, J=12.8, 3.6 Hz, 2H), 2.04 (m, 4H), 1.82 (dt, J=12.8, 3.6 Hz, 2H)1.40 (m, 2H), 1.17 (m, 2H); ¹³C-NMR (100 MHz, CDCl₃) δ 179.2, 154.2,144.1, 141.6, 138.3, 128.0, 127.3, 125.2, 120.3, 115.2, 66.5, 64.1,47.5, 35.2, 33.6, 23.5; HRMS (ESI) m/z for C₂₇H₃₁NO₄ [M+H]⁺ calcd434.2331. found 434.2334.

Ring Closing Metathesis of Fmoc-Protected Bis-Pentenyl Glycine EthylEster 12.

A solution of Fmoc-protected bis-pentenyl glycine ethyl ester 12 (116mg, 0.25 mmol) in 1,2-dichloroethane (degassed, 50 mL for 0.005M) wasstirred in the presence of Grubbs catalyst 1^(st) generation (41 mg,0.05 mmol, 20 mol %) at room temperature. After 19 hours, LC/MS datafrom the reaction mixture showed that only 5% of unreacted startingmaterial was left and that at least five different isomers of dimericcyclized product 15 were formed. Presence of monomeric cyclized product13 was not detected. Intermediate 14 was not detected, indicating thesecond metathesis (intramolecular RCM) might have proceeded rapidly.After the solvent was removed under reduced pressure, the products werepurified by silica gel column chromatography (eluting with 12.5% ethylacetate in n-hexanes) as a white foam: ¹H-NMR (400 MHz, CDCl₃) δ7.78-7.75 (m, 4H), 7.65-7.61 (m, 4H), 7.42-7.37 (m, 4H), 7.34-7.29 (m,4H), 6.01 (br s, 0.6H), 5.95 (br s, 0.3H), 5.92 (br s, 1.1H), 5.19-5.11(m, 4H), 4.39 (d, J=7.2 Hz, 4H), 2.47-2.41 (m, 2H), 2.26-2.20 (m, 4H),2.06-1.66 (m, 10H), 1.54-1.31 (m, 10H), 1.04-0.76 (m, 4H); HRMS (ESI)m/z for C₅₄H₆₆N₃O₈ [M+NH₄]⁺ calcd 884.4850. found 884.4857.

Peptide Synthesis.

The peptides were prepared using Fmoc chemistry on Rink Amide MBHA resin(NovaBiochem) with a loading capacity of 0.66 mmol/g. The dry resin wasswelled with 1-methyl-2-pyrrolidinone (NMP) for 15 min before use. TheFmoc protecting group was removed by treatment with 25% piperidine inNMP (3×5 min). Natural amino acids were coupled for 30 min using2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU) as the activating agent, 10 equivalents ofFmoc-protected amino acid, and 20 equivalents of diisopropyl ethylamine(DIPEA) in NMP. For the coupling of unnatural olefin-bearing aminoacids, a reaction time of 2 hours was used with 4 equivalents of aminoacid and 8 equivalents of DIPEA. After each coupling or deprotectingreaction, the resin was washed with NMP (3×3 min), CH₂Cl₂ (5×3 min), andNMP (3×3 min). After the final Fmoc deprotection, the free N-terminuswas acetylated by treatment with 30 equivalents of acetic anhydride and60 equivalents of DIPEA in NMP for 2 hours.

Metathesis and Purification.

Ring closing metathesis of resin-bound N-terminal capped peptides wasperformed using 20 mol % Grubbs catalyst in degassed 1,2-dichloroethane(DCE) for 2 hours at room temperature. When metathesis was incomplete,the reaction solution was drained and the resin was treated with freshcatalyst for another 2 hours. The resin was washed with DCE (5×3 min),CH₂Cl₂ (5×3 min), and methanol (3×3 min) and then dried in vacuoovernight. The peptides were cleaved from the resin by treatment with amixture of trifluoroacetic acid/triisopropylsilane/water (95/2.5/2.5)for 2 hours and precipitated by addition of cold diethyl ether. Theprecipitate was collected by centrifugation and washed twice with colddiethyl ether. The crude peptides were dissolved in methanol, filteredto remove resin, and purified by reverse phase HPLC to give pure peptideproducts.

Electrospray Ionization Mass Spectrometry (ESI-MS).

Peptide 9. ESIMS for C₇₅H₁₁₁N₂₀O₂₁ [M+H]⁺ calcd 1627.8. found 1627.6.

Peptide 4. ESIMS for C₉₁H₁₃₇N₂₀O₁₉ [M+H]⁺ calcd 1814.0. found 1814.0.

Peptide 6. ESIMS for C₈₂H₁₂₃N₂₀O₁₉ [M+H]⁺ calcd 1691.9. found 1691.6.

Peptide 5. ESIMS for C₈₅H₁₂₉N₂₀O₁₉ [M+H]⁺ calcd 1734.0. found 1734.0.

Peptide 8. ESIMS for C₈₈H₁₃₁N₂₀O₁₉ [M+H]⁺ calcd 1772.0. found 1772.0.

Circular Dichroism.

Peptides were dissolved in water to described concentrations, and theconcentrations were determined by absorbance spectroscopy (extinctioncoefficient for tryptophan, ε₂₈₀=5690 cm⁻¹). Circular dichroism spectrawere collected on a Jasco J-710 spectropolarimeter equipped with atemperature controller using the following standard measurementparameters: 0.5 nm step resolution, 20 nm/sec speed, 10 accumulations, 1sec response, 1 nm bandwidth, 0.1 cm path length. All spectra wereconverted to a uniform scale of molar ellipticity after backgroundsubtraction. Temperature-dependent CD spectra of each peptide (94-100μM) were recorded at varying temperatures (4° C. and every 10° C. from10° C. to 90° C.) from 260 to 185 nm. CD measurements with varyingconcentrations (18, 48, 70, and 118 μM) of peptide 4 were performed at20° C. To generate thermal unfolding curves, the ellipticity at 222 nmfor each peptide (94-100 μM) was measured every 1° C. from 4 to 95° C.with temperature slope of 3° C./min. To obtain T_(m), we analyzed thethermal unfolding curves using a two-state model as previously describedwith 95% confidence interval (see Favrin, G.; Irback, A.; Samuelsson,B.; Wallin, S. Biophysic. J. 2003, 85, 1457-1465). Stitched peptides 4and 8 did not have a cooperative melting transition point in thistemperature range, and therefore their T_(m) could not determined bythis method. However, peptide 4 retained more than 50% of theiralpha-helicity even at 95° C.

Peptide Digestion Assay.

0.4 mL of trypsin immobilized on agarose (Pierce, catalog #20230) waswashed with 0.8 mL of a digestion buffer (0.1 M NH₄HCO₃ buffer, pH 8.0).The gel was separated from the buffer after each wash by centrifugation.The washed enzyme was suspended in 1.6 mL of the digestion buffer. 350μL of a peptide solution (24 μM) in the digestion buffer was mixed with150 μL of the enzyme suspension and the resulting mixture was incubatedwith rapid shaking at room temperature for 10, 30, 90, 135, 180 minutes.The incubation was quenched by filtering off the enzyme, and theresidual substrate in the filtrate was quantified by HPLC-based peakdetection at 280 nm. The digestion assay displayed first order kinetics.The half-life, t_(1/2), was determined by linear regression analysisusing Kaleida graph (Synergy Software) from a plot of ln[S] versus time(min) (t_(1/2)=ln 2/slope, slope: 4.04±0.16×10⁻⁵ min⁻¹(4);7.11±0.66×10⁻⁵ min⁻¹ (5)).

Molecular Modeling Study.

A Monte Carlo conformational search was performed to locate all lowenergy conformations of each linker in the helical state. To generatestarting conformations for the MC conformational search, a 15-residuepolyalanine peptide was built with a right-handed helical conformationusing MacroModel's Maestro GUI (Macromodel, v.9.1, Schrodinger, LLC, NewYork, N.Y., 2005). Hydrocarbon cross-links were manually added, and werefully minimized while all non-cross-linker atoms were held frozen. Foreach isomer, two distinct 10,000 step Monte Carlo conformationalsearches were run. For all calculations, energies were evaluated usingthe OPLS2005 force field, as implemented in Macromodel (Macromodel,v.9.1, Schrodinger, LLC, New York, N.Y., 2005). For all minimizationsthe Polak-Ribiere Conjugate Gradient (PRCG) method was employed, and theconvergence criterion for the minimization of gradient norm was set to<0.05 kJ/mol-Å. We employed the GB/SA solvation treatment (Still, W. C.;Tempczyk, A.; Hawlely, R. C.; Hendrickson, T. A., A General Treatment ofSolvation for Molecular Mechanics. J. Am. Chem. Soc. 1990, 112,6127-6129.), modeling the solvent as chloroform as all metathesisreactions were carried out in 1,2-dichloroethane. Bond dipole cutoffswere employed to truncate the electrostatic and GB terms. Non-bondedcutoffs were as follows: 8 Å in Van der Waals, 99999.0 Å incharge-charge (effectively infinite), 20 3/2 A (89.4 Å) incharge-dipole, and 20 Å in dipole-dipole. Harmonic constraints (100kJ/mol) were placed on each backbone dihedral angle to maintain thehelical conformation throughout the search. At each step of the MonteCarlo search, 2-5 cross-linker dihedrals were randomly selected, andtheir values were adjusted by 0-180°. The C-terminal C—C bond adjacentto each olefin was temporarily broken during each step—allowing fordihedral perturbations along the cross-linker—and then reattached afterdihedral modification. After each step up to 500 steps of minimizationwere performed—if convergence was not achieved in less steps—andconformations within 50 kJ of the global minimum were saved. After thesearch, all remaining structures were fully minimized, and allconformations within 15 kJ of the global minimum were kept, whileredundant structures (RMSD<0.25 Å) were removed. The number of newstructures obtained after pooling the conformations obtained from thesecond run with those obtained from the first run was insignificant,suggesting that conformational space had been fully explored.

Molecular Modeling Study of i,i+4,i+4+7 System (Peptide 4 Versus 3).

Molecular modeling suggests that the lowest energy double bond isomer ofproduct 4 is lower in energy than the most stable isomer of 3 by −15kcal/mol (Table 6). This is in part due to three syn-pentaneinteractions that arise in product 3: two are located at the spirojunction while one is located at the N-terminal attachment of the staple(FIGS. 11, C and D). In the product 4, we also see a preference of ˜2.5kcal/mol for a cis double bond in the i,i+4 staple. Although there is noapparent enthalpic preference for either double bond orientation in thei,i+7 staple, the cis double bond seems to be entropically favored,since there are more low energy states present for this isomer (31versus 18, Table 6).

TABLE 6 Energy (kcal/mol)^(a) Conformations^(b) i, i + 4, i + 4 + 7Peptide 4 Peptide 3 Peptide 4 Peptide 3 cis/cis 0.1 (−466.4) 15.3(−451.2) 31 25 cis/trans 0.0 (−466.5) 15.8 (−450.7) 18 61 trans/cis 2.5(−464.0) 14.9 (−451.6) 16 32 trans/trans 2.4 (−464.1) 15.0 (−451.5) 9 45^(a)Energy is that of global minimum relative to global minimum oflowest energy isomer; absolute energies are reported in parenthesis.^(b)The number of conformations located within 15 kJ/mol (3.59 kcal) ofthe global minimum of each isomer.

Molecular Modeling Study of i,i+4,i+4+4 System (Peptide 8 Versus 16).

Molecular modeling suggests that the lowest energy double bond isomer ofproduct 8 is lower in energy than the most stable isomer of 16 by ˜14kcal/mol (Table 7). This is in part due to four syn-pentane interactionsthat are present in product 16: two are located at the spiro junctionwhile one is located at each of the terminal attachments of thecrosslink to the peptide backbone (FIGS. 12, C and D). We see that thecis/cis isomer of product 8 is the most energetically favorable one. Theaddition of a trans double bond in the i,i+4 linkage is unfavorable by˜2 kcal, while substituting the cis for a trans double bond in thei+4,i+4+4 staple costs ˜6 kcal. Interestingly, the lowest energy isomerfor the product 16 is the trans/trans isomer. Adding an N-terminal cisbond costs ˜0.5 kcal, while making this substitution on the C-terminallinkage is disfavored by ˜1.5 kcal.

TABLE 7 Energy (kcal/mol)^(a) Conformations^(b) i, i + 4, i + 4 + 4Peptide 8 Peptide 16 Peptide 8 Peptide 16 cis/cis 0.0 (−462.0) 15.6(−446.3) 4 16 cis/trans 6.1 (−455.9) 14.1 (−447.9) 12 8 trans/cis 2.3(−459.7) 15.0 (−446.9) 8 17 trans/trans 8.0 (−453.9) 13.5 (−448.5) 19 8^(a)Energy is that of global minimum relative to global minimum oflowest energy isomer; absolute energies are reported in parenthesis.^(b)The number of conformations located within 15 kJ/mol (3.59 kcal) ofthe global minimum of each isomer.

Example of Multiple-Stitching.

To investigate the possibility of peptides stabilized by three and morecrosslinks, peptide 17 (FIG. 13) was designed to contain S₅ at i, two B₅at i+4 and i+8, and S₅ at i+12 on solid support and subjected it toring-closing metathesis using 30% Grubbs catalyst in dichloroethanesolvent. Small portions of the peptide-containing resin were taken outfrom the reaction vessel at the time indicated (FIG. 14), and theproducts were analyzed by LCMS after cleavage. LCMS results clearly showthe formation of single- and double-stapled intermediates, most of whichwere eventually consumed. A single product peak accounted for 90% ofproduct mixture, which had the molecular mass expected of the product oftriple crosslinking (peptide 24). A model peptide bearing B₅ at i andi+4 (peptide 25 in FIG. 15) did not produce double stapled compound 27providing only single stapled product 26. In addition, a model peptidecontaining R₅ at i and S₅ at i+4 position (peptide 28) did not undergoRCM to produce peptide 29 (FIG. 15). The results from this model studyindicated that peptide 24, as depicted in FIG. 13, to be the most likelystructure for the triple crosslinked product. This result suggest thatfour or more crosslinks also might be introduced to peptide system byrational design.

Example 2. Additional Stitched Peptides

Additional Stitched Peptides I: Other RNases A Analogs

TABLE 8 Peptide Ac-

WAETAAB ₅KFL

AHA-NH₂ Ia: (SEQ ID 12) [ESIMS for C₉₁H₁₃₈N₂₀O₁₉ [M/2 +H]⁺ calcd 907.5, found 907.6] Peptide Ac-

WAETAAB ₅KFLAAH

-NH₂ Ib: (SEQ ID 13) [ESIMS for C₉₄H₁₄₄N₂₀O₁₉ [M/2 +H]⁺ calcd 928.5, found 928.4] Peptide Ac-EWA

TAAB ₅KFL

AHA-NH₂ Ic: (SEQ ID 14) [ESIMS for C₈₈H₁₃₂N₂₀O₁₉ [M/2 +H]⁺ calcd 886.5, found 886.4] Peptide Ac-

EWAB ₅TAAB ₅KFL

AHA-NH₂ Id: (SEQ ID 15) [ESIMS for C₉₈H₁₄₇N₂₁O₂₀ [M/2 +H]⁺ calcd 969.1, found 968.8] Peptide Ac-linker1-EWA

TAAB ₅KFLAAH

-NH₂ Ie: (SEQ ID 16) [ESIMS for C₁₀₁H₁₅₆N₂₂O₂₃ [M/2 +H]⁺ calcd 1022.6, found 1022.4] Peptide Ac-linker1-

WAETAAB ₅KFLAAH

-NH₂ If: (SEQ ID 17) [ESIMS for C₁₀₄H₁₆₂N₂₂O₂₃ [M/2 +H]⁺ calcd 1043.6, found 1043.2] Peptide Ac-linker1-EWA

TAAB ₅KFL

AHA-NH₂ Ig: (SEQ ID 18) [ESIMS for C₉₈H₁₅₀N₂₂O₂₃ [M/2 +H]⁺ calcd 1001.6, found 1001.2] Peptide FITC-linker1-

WAETAAB ₅KFLAAH

-NH₂ Ih: (SEQ ID 19) [ESIMS for C₁₂₃H₁₇₂N₂₃O₂₇S [M/3 +H]⁺ calcd 811.7, found 811.6] Peptide FITC-linker1-EWA

TAAB ₅KFL

AHA-NH₂ Ii: (SEQ ID 20) [ESIMS for C₁₁₇H₁₆₀N₂₃O₂₇S [M/3 +H]⁺ calcd 783.7, found 783.6]

Additional Stitched Peptides II: FITC-Labeled RNases a Analogs

TABLE 9 Peptide FITC-linker1-EWAETAAAKFLAAHA-NH₂ IIa:(SEQ ID 21) [ESIMS for C₁₀₄H₁₃₉N₂₃O₂₉S [M/2 +H]⁺ calcd 1102.5, found 1102.8] Peptide FITC-linker1-EWA

TAA

KFLAAH

-NH₂ IIb (SEQ ID 22) [ESIMS for C₁₁₁H₁₅₁N₂₃O₂₇S [M/2 +H]⁺ calcd 1135.0, found 1134.8] Peptide FITC-linker1-EWA

TAA

KFLAAH

-NH₂ IIc: (SEQ ID 23) [ESIMS for C₁₁₁H₁₅₁N₂₃O₂₇S [M/2 +H]⁺ calcd 1135.0, found 1134.8] Peptide FITC-linker1-EWA

TAA

KFLAAH

-NH₂ IId: (SEQ ID 24) [ESIMS for C₁₁₄H₁₅₇N₂₃O₂₇S [M/2 +H]⁺ calcd 1156.1, found 1155.6] Peptide FITC-linker1-EWA

TAAB ₅KFLAAH

-NH₂ IIe: (SEQ ID 25) [ESIMS for C¹²⁰H¹⁶⁵N²³O²⁷S [M/2 +H]⁺ calcd 1196.1, found 1195.6]

Additional Stitched Peptides III: Hydrophilic stitched peptide analogs

TABLE 10 Peptide Ac-EWS

TDN

KQEADR

-NH₂ IIIa: (SEQ ID 26) [ESIMS for C₇₄H₁₁₇N₂₃O₂₈ [M/2 +H]⁺ calcd 887.4, found 888.0] Peptide Ac-EWS

TDNB ₅KQEADR

-NH₂ IIIb: (SEQ ID 27) [ESIMS for C₈₉H₁₃₉N₂₃O₂₈ [M/2 +H]⁺ calcd 989.0, found 989.2] Peptide Ac-EWS

TDNB ₅KQE

DRA-NH₂ IIIc: (SEQ ID 28) [ESIMS for C₈₆H₁₃₃N₂₃O₂₈ [M/2 +H]⁺ calcd 968.0, found 968.4]

Additional Stitched Peptides IV: Rev-Based Peptides Targeting HIV-RRE

TABLE 11 Peptide Ac-TRQ

RRNB ₅RRRWRE

QR-NH₂ IVa: (SEQ ID 29) [ESIMS for C₁₁₁H₁₉₃N₄₆O₂₄ [M/3 +H]⁺ calcd 851.5, found 852.0] Peptide Ac-TRQ

RRNB ₅WRR

RERQR-NH₂ IVb: (SEQ ID 30) [ESIMS for C₁₀₈H₁₈₇N₄₆O₂₄ [M/3 +H]⁺ calcd 837.5, found 837.9] Peptide FITC-linker2-TRQ

RRNB ₅RRRWRE

QR-NH₂ IVc: (SEQ ID 31) [ESIMS for C₁₃₃H₂₀₇N₄₈O₂₉ [M/3 +H]⁺ calcd 990.9, found 991.2] Peptide FITC-linker2-TRQ

RRNB ₅WRR

RERQR-NH₂ IVd: (SEQ ID 32) [ESIMS for C₁₃₀H₂₀₁N₄₈O₂₉ [M/3 +H]⁺ calcd 976.8, found 977.2]

Additional Stitched Peptides V: ARNT-Based Peptides Targeting HIF-1α

TABLE 12 Peptide Ac-IL

MAVB ₅HMKSLR

T-NH₂ Va: (SEQ ID 33) [ESIMS for C₉₀H₁₅₈N₂₂O₁₈S₂ [M/2 +H]⁺ calcd 949.6, found 950.0] Peptide Ac-ILRMAV

HMKB ₅LRG

-NH₂ Vb: (SEQ ID 34) [ESIMS for C₈₈H₁₅₅N₂₅O₁₆S₂ [M/2 +H]⁺ calcd 941.1, found 941.6] Peptide FITC-linker2-IL

MAVB ₅HMKSLR

T-NH₂ Vc: (SEQ ID 35) Peptide FITC-linker2-ILRMAV

HMKB ₅LRGR-NH₂ Vd: (SEQ ID 36)

Additional Stitched Peptides VI: p53-Based Peptides Targeting hDM-2 andhDMx

TABLE 13 Peptide Ac-LS

ETFB ₅DLWKLL

EN-NH₂ VIa: (SEQ ID 37) [ESIMS for C₁₀₄H₁₆₂N₂₀O₂₆ [M/2 +H]⁺ calcd 1053.6, found 1054.0] Peptide Ac-LS

ETAB ₅DLWKLL

EN-NH₂ VIb: (SEQ ID 38) [ESIMS for C₉₈H₁₅₈N₂₀O₂₆ [M/2 +H]⁺ calcd 1015.6, found 1016.0] Peptide FITC-linker2-LS

ETFB ₅DLWKLL

EN-NH₂ VIc: (SEQ ID 39) [ESIMS for C₁₂₆H₁₇₆N₂₂O₃₁S [M/2 +H]⁺ calcd 1262.6, found 1262.8] Peptide FITC-linker2-LS

ETAB ₅DLWKLL

EN-NH₂ VId: (SEQ ID 40) [ESIMS for C₁₂₂H₁₇₂N₂₂O₃₁S [M/2 +H]⁺ calcd 1224.6, found 1224.8] Peptide Biotin-linker1-LS

ETFB ₅DLWKLL

EN-NH₂ VIe: (SEQ ID 41) [ESIMS for C₁₂₂H₁₉₂N₂₄O₃₁S [M/2 +H]⁺ calcd 1260.7, found 1261.2] Peptide Biotin-linker1-LS

ETAB ₅DLWKLL

EN-NH₂ VIf: (SEQ ID 42) [ESIMS for C₁₁₆H₁₈₈N₂₄O₃₁S [M/2 +H]⁺ calcd 1222.7, found 1222.8] Peptide FITC-linker2-

DFSB ₅YWK

L-NH₂ VIg: (SEQ ID 43) [ESIMS for C₉₆H₁₁₉N₁₅O₂₀S [M/2 +H]⁺ calcd 916.9, found 917.2] Peptide FITC-linker2-

DFSB ₅YWK

L-NH₂ VIh: (SEQ ID 44) [ESIMS for C₉₆H₁₁₉N₁₅O₂₀S [M/2 +H]⁺ calcd 916.9, found 917.6]

Additional Stitched Peptides VII: BID-BH3-Based Peptides TargetingBCL-X_(L)

TABLE 14 Peptide Ac-EDIIRNIA

HLAB ₅VGDWN_(L)D

SI-NH₂ VIIa: (SEQ ID 45) [ESIMS for C₁₁₇H₁₈₅N₂₉O₃₂ [M/2 +H]⁺ calcd 1324.7, found 1325.2] Peptide Ac-NIA

HLAB ₅VGDWN_(L)D

SI-NH₂ VIIb: (SEQ ID 46) [ESIMS for C₉₀H₁₃₉N₂₁O₂₃ [M/2 +H]⁺ calcd 1011.58, found 1012.0] Peptide Ac-NIA

HLAB ₅VGDWN_(L)D

-NH₂ VIIc: (SEQ ID 47) [ESIMS for C₈₁H₁₂₁N₁₉O₂₀ [M/2 +H]⁺ calcd 911.5, found 912.0] Peptide FITC-linker2-EDIIRNIA

HLAB ₅VGDWN_(L)D

SI-NH₂  VIId: (SEQ ID 48) [ESIMS for C₁₃₉H₁₉₉N₃₁O₃₇S [M/2 +H]⁺ calcd 1533.8, found 1534.4] Peptide FITC-linker2-NIA

HLAB ₅VGDWN_(L)D

SI-NH₂ VIIe: (SEQ ID 49) [ESIMS for C₁₁₂H₁₅₃N₂₃O₂₈S [M/2 +H]⁺ calcd 1220.6, found 1221.2] Peptide FITC-linker2-NIA

HLAB ₅VGDWN_(L)D

-NH₂ VIIf: (SEQ ID 50) [ESIMS for C₁₀₃H₁₃₇N₂₁O₂₅S [M/2 +H]⁺ calcd 1120.6, found 1120.8] N_(L) = norleucine

Additional Stitched Peptides VIII: hE47-Based Peptides Targeting IdProteins

TABLE 15 Peptide Ac-L

ILQB ₅AVQ

ILGLEQQVRER-NH₂ VIIIa: (SEQ ID 51) [ESIMS for C₁₁₆H₁₉₉N₃₁O₂₉ [M/3 +H]⁺ calcd 854.9, found 855.2] Peptide Ac-L

ILQB ₅AVQVIL

LEQQVRER-NH₂  VIIIb: (SEQ ID 52) [ESIMS for C₁₂₂H₂₁₁N₃₁O₂₉ [M/3 +H]⁺ calcd 882.9, found 883.2] Peptide Ac-LLILQQAV

VILB ₅LEQ

VRER-NH₂ VIIIc: (SEQ ID 53) [ESIMS for C₁₂₀H₂₁₁N₃₀O₂₈ [M/3 +H]⁺ calcd 863.9, found 864.0] Peptide Ac-LLILQQAV

VILB ₅LEQQVR

R-NH₂ VIIId: (SEQ ID 54) [ESIMS for C₁₂₃H₂₁₈N₃₁O₂₇ [M/3 +H]⁺ calcd 877.6, found 877.6] Peptide Ac-LLIL

QAVB ₅VIL

LEQQVRER-NH₂ VIIIe: (SEQ ID 55) [ESIMS for C₁₂₀H₂₁₁N₃₀O₂₈ [M/3 +H]⁺ calcd 863.9, found 864.4] Peptide Ac-LLIL

QAVB ₅VILGLE

QVRER-NH₂  VIIIf: (SEQ ID 56) [ESIMS for C₁₂₀H₂₁₁N₂₉O₂₇ [M/3 +H]⁺ calcd 854.2, found 854.4] Peptide Ac-LLIL

QAVB ₅VILB ₅LEQ

VRER-NH₂ VIIIg: (SEQ ID 57) [ESIMS for C₁₂₅H₂₁₅N₂₉O₂₇ [M/3 +H]⁺ calcd 876.2, found 876.4] Peptide FITC-linker2-L

ILQB ₅AVQ

ILGLEQQVRER-NH₂ VIIIh: (SEQ ID 58) [ESIMS for C₁₃₈H₂₁₆N₃₃O₃₄S [M/3 +H]⁺ calcd 994.2, found 994.5] Peptide FITC-linker2-L

ILQB ₅AVQVIL

LEQQVRER-NH₂ VIIIi: (SEQ ID 59) [ESIMS for C₁₄₄H₂₂₈N₃₃O₃₄S [M/3 +H]⁺ calcd 1022.2, found 1022.4] Peptide FITC-linker2-LLILQQAV

VILB ₅LEQ

VRER-NH₂ VIIIj: (SEQ ID 60) [ESIMS for C₁₄₂H₂₂₅N₃₂O₃₃S [M/3 +H]⁺ calcd 1003.2, found 1003.6] Peptide FITC-linker2-LLILQQAV

VILB ₅LEQQVR

R-NH₂ VIIIk: (SEQ ID 61) [ESIMS for C₁₄₅H₂₃₂N₃₃O₃₂S [M/3 +H]⁺ calcd 1016.9, found 1017.2] Peptide FITC-linker2-LLIL

QAVB ₅VIL

LEQQVRER-NH₂ VIIIl: (SEQ ID 62) [ESIMS for C₁₄₂H₂₂₅N₃₂O₃₃S [M/3 +H]⁺ calcd 1003.2, found 1003.6] Peptide FITC-linker2-LLIL

QAVB ₅VILGLE

QVRER-NH₂ VIIIm: (SEQ ID 63) [ESIMS for C₁₄₂H₂₂₆N₃₁O₃₂S [M/3 +H]⁺ calcd 993.6 found 994.0] Peptide FITC-linker2-LLIL

QAVB ₅VILB ₅LEQ

VRER-NH₂ VIIIn: (SEQ ID 64) [ESIMS for C₁₄₇H₂₃₂N₃₁O₃₂S [M/3 +H]⁺ calcd 1024.9, found 1015.6]

Additional Stitched Peptides IX: GLP-1-Based Peptides Targeting GLP-1Receptor

TABLE 16 Peptide HAEGTFTSDVSSY

EGQB ₅AKEB ₅IAW

VKGR-NH₂  IXa: (SEQ ID 65) [ESIMS for C₁₅₉H₂₄₅N₄₀O₄₅ [M/3 +H]⁺ calcd 1144.94, found 1145.1] Peptide HAEGTFTSDVSSY

EGQB ₅AKEFIA

LVKGR-NH₂ IXb: (SEQ ID 66) [ESIMS for C₁₅₆H₂₄₆N₃₉O₄₅ [M/3 +H]⁺ calcd 1128.6, found 1128.8] Peptide HAEGTFTSDVSSYLEGQ

AKEB ₅IAWLVK

R-NH₂ IXc: (SEQ ID 67) [ESIMS for C₁₆₂H₂₅₅N₄₀O₄₅ [M/3 +H]⁺ calcd 1160.3, found 1160.8] Peptide HAEGTFTSDVSSYLEG

AAKB ₅FIAB ₅LVK

R-NH₂  IXd: (SEQ ID 68) [ESIMS for C₁₆₀H₂₅₃N₃₈O₄₂ [M/3 +H]⁺ calcd 1126.3, found 1126.4] Peptide HAEGTFTSDVSSYLEGQAAK

FIAB ₅LVK

R-NH2 IXe: (SEQ ID 69) [ESIMS for C₁₅₅H₂₄₆N₃₉O₄₃ [M/3 +H]⁺ calcd 1113.9, found 1114.4] Peptide HAEGTFTSD

SSYLEGB ₅AAKEFI

WLVKGR-NH₂ IXf: (SEQ ID 70) [ESIMS for C₁₆₆H₂₅₆N₃₉O₄₄ [M/3 +H]⁺ calcd 1166.6, found 1166.4] Peptide HAEGTFTSDVSSYLE

QAAB ₅EFIAWL

KGR-NH₂ IXg: (SEQ ID 71) [ESIMS for C₁₆₃H₂₄₈N₃₉O₄₅ [M/3 +H]⁺ calcd 1157.2, found 1156.8] Peptide HAEGTFTSDVSS

LEGQAAB ₅EFIAWL

KGR-NH₂ IXh: (SEQ ID 72) [ESIMS for C₁₅₉H₂₄₈N₃₉O₄₄ [M/3 +H]⁺ calcd 1135.9, found 1135.6] Peptide HAEGTFTSDVS

YLEB ₅QAAKEF

AWLVKGR-NH₂ IXi: (SEQ ID 73) [ESIMS for C₁₆₅H₂₅₃N₄₀O₄₄ [M/3 +H]⁺ calcd 1166.3, found 1166.0] Peptide HAEGTFTSD

SSYB ₅EGQAAK

FIAWLVKGR-NH₂ IXj: (SEQ ID 74) [ESIMS for C₁₆₀H₂₄₅N₄₀O₄₃ [M/3 +H]⁺ calcd 1138.3, found 1138.0] Peptide HAEGTFT

DVSB ₅YLEGQA

KEFIAWLVKGR-NH₂ IXk: (SEQ ID 75) [ESIMS for C₁₆₇H₂₅₇N₄₀O₄₃ [M/3 +H]⁺ calcd 1170.3, found 1170.0] Peptide HAEGTFT

DVSB ₅YLE

QAAKEFIAWLVKGR-NH₂ IXl: (SEQ ID 76) [ESIMS for C₁₆₅H₂₅₃N₄₀O₄₃ [M/3 +H]⁺ calcd 1161.0, found 1160.8] Peptide HAEGTFT

DVSB ₅YLEB ₅QAA

EFIAWLVKGR-NH₂ IXm: (SEQ ID 77) [ESIMS for C₁₆₉H₂₅₆N₃₉O₄₃ [M/3 +H]⁺ calcd 1173.3, found 1173.2] Peptide HAEGTFTSDVS

YLEB ₅QAA

EFIAWLVKGR-NH₂ IXn: (SEQ ID 78) [ESIMS for C₁₆₂H₂₄₆N₃₉O₄₄ [M/3 +H]⁺ calcd 1147.3, found 1146.8]

Additional Stitched Peptides X: NS5A-based peptides targeting HepatitisC Virus

TABLE 17 Peptide SGSWLRD

WDWB ₅CTVLTD

KTWLQSKL-NH₂ Xa: (SEQ ID 79) [ESIMS for C₁₆₁H₂₄₅N₃₈O₄₀S [M/3 +H]⁺ calcd 1127.6, found 1127.6] Peptide SGSWLRDVWDWI

TVLB ₅DFKB ₅WLQ

KL-NH₂  Xb: (SEQ ID 80) [ESIMS for C₁₇₄H₂₅₉N₃₈O₃₇ [M/3 +H]⁺ calcd 1157.7, found 1157.6] Peptide SGSWL

DVWB ₅WICTVL

DFKTWLQSKL-NH₂ Xc: (SEQ ID 81) [ESIMS for C₁₆₇H₂₅₀N₃₅O₃₇S [M/3 +H]⁺ calcd 1123.3, found 1123.6] Peptide SGSWL

DVWB ₅WIC

VLTDFKTWLQSKL-NH₂ Xd: (SEQ ID 82) [ESIMS for C₁₆₄H₂₄₄N₃₅O₃₇S [M/3 +H]⁺ calcd 1109.3, found 1109.2] Peptide SGSWL

DVWB ₅WICB ₅VLT

FKTWLQSKL-NH₂ Xe: (SEQ ID 83) [ESIMS for C₁₇₀H₂₅₄N₃₅O₃₅S [M/3 +H]⁺ calcd 1126.0, found 1126.0] Peptide SGSWLRDVW

WICB ₅VLTDFK

WLQSKL-NH₂ Xf: (SEQ ID 84) [ESIMS for C₁₆₉H₂₅₅N₃₈O₃₆S [M/3 +H]⁺ calcd 1141.7, found 1141.6] Peptide SGSWLRDVW

WICB ₅VLT

FKTWLQSKL-NH₂ Xg: (SEQ ID 85) [ESIMS for C₁₆₆H₂₄₈N₃₈O₃₅S [M/3 +H]⁺ calcd 1123.0, found 1122.8] Peptide SGSWLR

VWDWICB ₅VLTDFK

WLQSKL-NH₂ Xh: (SEQ ID 86) [ESIMS for C₁₇₂H₂₆₁N₃₈O₃₆S [M/3 +H]⁺ calcd 1155.7, found 1155.6] Peptide Ac-SGSWLRD

WDWB ₅CTVLTD

KTWLQSKL-NH₂ Xi: (SEQ ID 87) [ESIMS for C₁₆₃H₂₄₇N₃₈O₄₁S [M/3 +H]⁺ calcd 1141.6, found 1141.6] Peptide Ac-SGSWLRDVWDWI

TVLB ₅DFKB ₅WLQ

KL-NH₂ Xj: (SEQ ID 88) [ESIMS for C₁₇₆H₂₆₁N₃₈O₃₈ [M/3 +H]⁺ calcd 1171.7, found 1171.6] Peptide Ac-SGSWL

DVWB ₅WICTVL

DFKTWLQSKL-NH₂ Xk: (SEQ ID 89) [ESIMS for C₁₆₉H₂₅₂N₃₅O₃₈S [M/3 +H]⁺ calcd 1137.3, found 1137.2] Peptide Ac-SGSWL

DVWB ₅WIC

VLTDFKTWLQSKL-NH₂ Xl: (SEQ ID 90) [ESIMS for C₁₆₆H₂₄₆N₃₅O₃₈S [M/3 +H]⁺ calcd 1123.3, found 1123.2] Peptide Ac-SGSWL

DVWB ₅WICB ₅VLT

FKTWLQSKL-NH₂ Xm: (SEQ ID 91) [ESIMS for C₁₇₂H₂₅₆N₃₅O₃₆S [M/3 +H]⁺ calcd 1140.0, found 1140.0] Peptide Ac-SGSWLRDVW

WICB ₅VLTDFK

WLQSKL-NH₂ Xn: (SEQ ID 92) [ESIMS for C₁₇₁H₂₅₇N₃₈O₃₇S [M/3 +H]⁺ calcd 1155.7, found 1155.6] Peptide Ac-SGSWLRDVW

WICB ₅VLT

FKTWLQSKL-NH₂ Xo: (SEQ ID 93) [ESIMS for C₁₆₈H₂₅₃N₃₈O₃₆S [M/3 +H]⁺ calcd 1137.0, found 1136.8] Peptide Ac-SGSWLR

VWDWICB ₅VLTDFK

WLQSKL-NH₂ Xp: (SEQ ID 94) [ESIMS for C₁₇₄H₂₆₃N₃₈0₃₇S [M/3 +H]⁺ calcd 1169.7, found 1169.6] Peptide Ac-linker1-SGSWLRD

WDWB ₅CTVLTD

KTWLQSKL- Xq: NH₂ (SEQ ID 95) [ESIMS for C₁₇₃H₂₆₅N₄₀O₄₅S  [M/3 +H]⁺ calcd 1218.7, found 1218.6] Peptide Ac-linker1-SGSWLRDVWDWI

TVLB ₅DFKB ₅WLQ

KL- Xr: NH₂ (SEQ ID 96) [ESIMS for C₁₈₆H₂₇₉N₄₀O₄₂ [M/3 +H]⁺ calcd 1248.7, found 1248.9] Peptide Ac-linker1-SGSWL

DVWB ₅WICTVL

DFKTWLQSKL- Xs: NH₂ (SEQ ID 97) [ESIMS for C₁₇₉H₂₇₀N₃₇O₄₂S [M/3 +H]⁺ calcd 1214.4, found 1214.4] Peptide Ac-linker1-SGSWL

DVWB ₅WIC

VLTDFKTWLQSKL- Xt: NH₂ (SEQ ID 98) [ESIMS for C₁₇₆H₂₆₄N₃₇O₄₂S [M/3 +H]⁺ calcd 1200.3, found 1200.3] Peptide Ac-linker1-SGSWL

DVWB ₅WICB ₅VLT

FKTWLQSKL- Xu: NH₂ (SEQ ID 99)  Peptide Ac-linker1-SGSWLRDVW

WICB ₅VLTDFK

WLQSKL- Xv: NH₂ (SEQ ID 100) [ESIMS for C₁₈₁H₂₇₅N₄₀O₄₁S [M/3 +H]⁺ calcd 1232.7, found 1232.7] Peptide Ac-linker1-SGSWLRDVW

WICB ₅VLT

FKTWLQSKL- Xw: NH₂ (SEQ ID 101) [ESIMS for C₁₇₈H₂₇₁N₄₀O₄₀S [M/3 +H]⁺ calcd 1214.0, found 1214.1] Peptide Ac-linker1-SGSWLR

VWDWICB ₅VLTDFK

WLQSKL- Xx: NH₂ (SEQ ID 102) [ESIMS for C₁₈₄H₂₈₁N₄₀O₄₁S [M/3 +H]⁺ calcd 1246.7, found 1246.5] Peptide FITC-linker1-SGSWLRD

WDWB ₅CTVLTD

KTWLQSKL- Xy: NH₂ (SEQ ID 103) [ESIMS for C₁₉₂H₂₇₄N₄₁O₄₉S₂ [M/3 +H]⁺ calcd 1334.4, found 1334.1] Peptide FITC-linker1-SGSWLRDVWDWI

TVLB ₅DFKB ₅WLQ

KL- Xz: NH₂ (SEQ ID 104) [ESIMS for C₂₀₅H₂₈₈N₄₁O₄₆S [M/3 +H]⁺ calcd 1364.4, found 1364.4] Peptide FITC-linker1-SGSWL

DVWB ₅WICTVL

DFKTWLQSKL- Xaa: NH₂ (SEQ ID 105) [ESIMS for C₁₉₈H₂₇₉N₃₈O₄₆S₂ [M/3 +H]⁺ calcd 1330.0, found 1330.2] Peptide FITC-linker1-SGSWL

DVWB ₅WIC

VLTDFKTWLQSKL- Xab: NH₂ (SEQ ID 106) [ESIMS for C₁₉₅H₂₇₃N₃₈O₄₆S₂ [M/3 +H]⁺ calcd 1316.0, found 1316.1] Peptide FITC-linker1-SGSWL

DVWB ₅WICB ₅VLT

FKTWLQSKL- Xac: NH₂ (SEQ ID 107) Peptide FITC-linker1-SGSWLRDVW

WICB ₅VLTDFK

WLQSKL- Xad: NH₂ (SEQ ID 108) [ESIMS for C₂₀₀H₂₈₁N₄₁O₄₅S₂ [M/3 +H]⁺ calcd 1348.4, found 1348.2] Peptide FITC-linker1-SGSWLRDVW

WICB ₅VLT

FKTWLQSKL- Xae: NH₂ (SEQ ID 109) [ESIMS for C₁₉₇H₂₈₀N₄₁O₄₄S₂ [M/3 +H]⁺ calcd 1329.7, found 1330.0] Peptide FITC-linker1-SGSWLR

VWDWICB ₅VLTDFK

WLQSKL- Xaf: NH₂ (SEQ ID 110) [ESIMS for C₂₀₃H₂₉₀N₄₁O₄₅S₂ [M/3 +H]⁺ calcd 1362.4, found 1362.4] Peptide Biotin-linker1-SGSWLRD

WDWB ₅CTVLTD

KTWL Xag: QSKL-NH₂ (SEQ ID 111) [ESIMS for  C₁₈₁H₂₇₇N₄₂O₄₆S₂ [M/3 +H]⁺ calcd 1279.7, found 1280.1] Peptide Biotin-linker1-SGSWLRDVWDWI

TVLB ₅DFKB ₅WL Xah: Q

KL-NH₂ (SEQ ID 112) [ESIMS for  C₁₉₄H₂₇₆N₄₂O₄₃S [M/3 +H]⁺ calcd 1309.7, found 1310.1] Peptide Biotin-linker1-SGSWL

DVWB ₅WICTVL

DFKTWL Xai: QSKL-NH₂ (SEQ ID 113) [ESIMS for C₁₈₇H₂₈₂N₃₉O₄₃S₂ [M/3 +H]⁺ calcd 1275.4, found 1275.6] Peptide Biotin-linker1-SGSWL

DVWB ₅WIC

VLTDFKTWL Xaj: QSKL-NH₂ (SEQ ID 114) [ESIMS for C₁₈₄H₂₇₆N₃₉O₄₃S₂ [M/3 +H]⁺ calcd 1261.4, found 1261.8] Peptide Biotin-linker1-SGSWL

DVWB ₅WICB ₅VLT

FKTWL Xak: QSKL-NH₂ (SEQ ID 115) Peptide Biotin-linker1-SGSWLRDVW

WICB ₅VLTDFK

WL Xal: QSKL-NH₂ (SEQ ID 116) [ESIMS for C₁₈₉H₂₈₇N₄₂O₄₂S₂ [M/3 +H]⁺ calcd 1293.7, found 1294.2] Peptide Biotin-linker1-SGSWLRDVW

WICB ₅VLT

FKTWL Xam: QSKL-NH₂ (SEQ ID 117) [ESIMS for C₁₈₆H₂₈₃N₄₂O₄₁S₂ [M/3 +H]⁺ calcd 1275.1, found 1275.3] Peptide Biotin-linker1-SGSWLR

VWDWICB ₅VLTDFK

WL Xan: QSKL-NH₂ (SEQ ID 118) [ESIMS for C₁₉₂H₂₉₃N₄₂O₄₂S₂ [M/3 +H]⁺ calcd 1307.7, found 1308.3]

Additional Stitched Peptides XI: Max-Based Peptides Targeting Myc

TABLE 18 Peptide Ac-KATEYIQYN_(L)

RKNB ₅THQQDI

DL-NH₂ XIa: (SEQ ID 119) [ESIMS for C₁₃₃H₂₁₂N₃₅O₃₈ [M/3 +H]⁺ calcd 992.6, found 992.6] Peptide Ac-KATEYI

YMARKNB ₅THQQDI

DL-NH₂ XIb: (SEQ ID 120) [ESIMS for C₁₃₇H₂₂₂N₃₇O₃₇ [M/3 +H]⁺ calcd 1015.9, found 1016.3]

Additional Stitched Peptides XII: MITF-Based Peptides Targeting MITF

TABLE 19 Peptide Ac-TILKASVDY

RKLB ₅REQQRA

EL-NH₂ XIIa: (SEQ ID 121) [ESIMS for C₁₂₈H₂₂₀N₃₅O₃₃ [M/3 +H]⁺ calcd 972.6, found 972.8] Peptide Ac-TILKAS

DYIRKLB ₅REQQRA

EL-NH₂ XIIb: (SEQ ID 122) [ESIMS for C₁₃₂H₂₂₈N₃₅O₃₃ [M/3 +H]⁺ calcd 991.3, found 991.4]

LISTING OF ABBREVIATIONS

TABLE 20* FITC

Biotin

linker1

linker2

Ac

R₅

R₈

S₅

S₈

B₅

Aib

Other Embodiments

The foregoing has been a description of certain non-limiting preferredembodiments of the invention. Those of ordinary skill in the art willappreciate that various changes and modifications to this descriptionmay be made without departing from the spirit or scope of the presentinvention, as defined in the following claims.

We claim:
 1. A method of preparing an alpha-helical polypeptide, saidmethod comprising the steps of: (i) providing an amino acid of Formula(A):

or a salt thereof; (ii) providing an amino acid of the Formula (B):

or a salt thereof; (iii) providing an amino acid of the Formula (C):

or a salt thereof; (iv) providing at least one additional amino acid;(v) coupling said amino acids of Formulae (A), (B), and (C) with atleast one amino acid of step (iv); and (vi) treating the polypeptide ofstep (v) with a catalyst; wherein each instance of L₁ and L₂ is,independently, a straight chain alkylene of 1 to 7 carbon atoms; K is astraight chain alkylene of 1 to 7 carbon atoms; M is a straight chainalkylene of 1 to 7 carbon atoms; each instance of R^(a) is,independently, hydrogen; cyclic or acyclic, branched or unbranched,substituted or unsubstituted aliphatic; cyclic or acyclic, branched orunbranched, substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; cyclic oracyclic, substituted or unsubstituted acyl; or an amino protectinggroup; R^(b) is hydrogen; cyclic or acyclic, branched or unbranched,substituted or unsubstituted aliphatic; each instance of R^(c), is,independently, hydrogen; cyclic or acyclic, branched or unbranched,substituted or unsubstituted aliphatic; cyclic or acyclic, branched orunbranched, substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; cyclic oracyclic, substituted or unsubstituted acyl; substituted or unsubstitutedhydroxyl; substituted or unsubstituted thiol; substituted orunsubstituted amino; cyano; isocyano; halo; or nitro; each instance ofR^(e) is, independently, —R^(E), wherein each instance of —R^(E) is,independently, hydrogen, cyclic or acyclic, branched or unbranched,substituted or unsubstituted aliphatic; cyclic or acyclic, branched orunbranched, substituted or unsubstituted heteroaliphatic; substituted orunsubstituted aryl; substituted or unsubstituted heteroaryl; substitutedor unsubstituted acyl; a resin; or a hydroxyl protecting group; eachinstance of R^(f) is, independently, hydrogen; cyclic or acyclic,branched or unbranched, substituted or unsubstituted aliphatic; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedheteroaliphatic; substituted or unsubstituted aryl; substituted orunsubstituted heteroaryl; substituted or unsubstituted acyl; a resin; anamino protecting group; or a label optionally joined by a linker,wherein the linker is selected from cyclic or acyclic, branched orunbranched, substituted or unsubstituted alkylene; cyclic or acyclic,branched or unbranched, substituted or unsubstituted alkenylene; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedalkynylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted heteroalkylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroalkenylene; cyclic or acyclic,branched or unbranched, substituted or unsubstituted heteroalkynylene;substituted or unsubstituted arylene; substituted or unsubstitutedheteroarylene; or substituted or unsubstituted acylene; or R^(f) andR^(a) together form a substituted or unsubstituted heterocyclic orheteroaromatic ring; and each instance of x is, independently, aninteger between 0 to 3, inclusive.
 2. The method of claim 1, whereinsaid catalyst is a ring closing metathesis catalyst.
 3. The method ofclaim 1, wherein said catalyst is a ruthenium catalyst.
 4. The method ofclaim 1, wherein each R^(a) is hydrogen or methyl.
 5. The method ofclaim 1, wherein each R^(a) is hydrogen.
 6. The method of claim 1,wherein at least one R^(a) is —COCH₃.
 7. The method of claim 1, whereineach W is alkyl.
 8. The method of claim 1, wherein each R^(b) is methyl.9. The method of claim 1, wherein each R^(f) is hydrogen.
 10. The methodof claim 1, wherein each R^(e) is hydrogen.
 11. The method of claim 1,wherein each R^(f) is an amino protecting group.
 12. The method of claim1, wherein each R^(f) is Boc.
 13. The method of claim 1, wherein eachR^(f) is Fmoc.
 14. The method of claim 1, wherein each

is a double bond.
 15. The method of claim 1, wherein the amino acid ofFormula (A) is an amino acid of Formula (A-2):


16. The method of claim 15, wherein the amino acid of Formula (A-2) isof one of the following formulae:


17. The method of claim 1, wherein an amino acids of Formulae (B) and(C) are independently of one of the following formulae:


18. The method of claim 1, wherein an amino acids of Formulae (B) and(C) are independently of one of the following formulae: